Rapid diagnostic device, assay and multifunctional buffer

ABSTRACT

A rapid diagnostic device, assay and multifunctional buffer reagent are provided for the detection of a target analyte in a fluid test sample. The 2-step assay utilizes a dual component flow-through device comprising a test unit and a dried indicator reagent delivery unit for receiving the fluid sample and multifunctional buffer, respectively. The test unit comprises a reaction zone containing immobilized capture reagent that can bind to the target analyte, an absorbent zone supporting the reaction zone, and optionally, a blood separation zone in lateral fluid communication with the reaction zone. The delivery unit comprises a label zone permeated with a dried indicator reagent which can be placed in transient fluid communication with the reaction zone of the test unit during the assay procedure. The rapid diagnostic assay system reduces the number of assay reagents, method steps and time required for performance compared to other conventional assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 12/285,868, filed Oct. 15,2008, which is a continuation of application Ser. No. 10/163,675, filedJun. 6, 2002, now U.S. Pat. No. 7,531,362 B2, which claims the benefitof Provisional Application Nos. 60/296,147 and 60/309,477, filed Jun. 7,2001 and Aug. 3, 2001, respectively, the entire contents of each ofwhich are hereby incorporated by reference in this application.

FIELD OF THE INVENTION

An improved rapid diagnostic device, assay and multifunctional bufferare provided for the detection of a target analyte in a fluid testsample. The assay utilizes a multifunctional buffer reagent andflow-through device comprising a test unit in combination with adetachable dried indicator reagent delivery unit. A method for utilizingthe flow-through device, a test kit and a formulation for generating themultifunctional buffer are also provided.

BACKGROUND OF THE INVENTION

Diagnostic assays have become an indispensable aid in medical andresearch fields for detecting a variety of components in biologicalfluids and tissue samples such as drugs, hormones, enzymes, proteins,antibodies, and infectious agents. A fundamental principle underlyingthe operation of a number of these assays is a specific recognition andbinding reaction that occurs between two or more members to form acomplex that can subsequently be detected. Normally, the members involvea capture reagent (e.g. receptor) that will specifically recognize andbind to a target analyte of interest (e.g. ligand) in a fluid testsample (e.g. whole blood, plasma, serum, urine, saliva, etc.). Moreover,a visually detectable indicator reagent is included in the reactionwhich will recognize and bind to any analyte complexed with the capturereagent to produce a signal indicating that a positive reaction hasoccurred. In particular, immunological assays are designed to functionon the basis of antibody recognition and selective binding reaction toantigen and accordingly, have proven extremely valuable over the yearsin clinical applications for the detection of numerous infectiousdisease states.

In order to achieve accurate results, however, immunoassays oftenrequire precision in performing a series of time-consuming steps, aswell as technical knowledge in operating sophisticated laboratoryequipment. Accordingly, their use in diagnosing infectious disease hasbeen essentially confined to clinical facilities that have the necessaryresources for making such determinations including highly trainedtechnical personnel and laboratories equipped with appropriatediagnostic equipment.

On this basis, as with many technologies, immunodiagnostic testing isevolving towards more simplistic approaches in the rapid identificationand diagnosis of infectious disease states. The need for a simplisticqualitative assay for detecting analyte in a biological sample isbecoming more desirable since it would offer an appealing possibilityfor use in less conventional settings having limited resources, e.g.physician's office, or domestic household. Whether in a public healthclinic or a rural setting, it is preferable that an assay for detectionof a target analyte in a fluid test sample be performed without the aidof complicated instruments and the requisite skills and knowledge ofprofessionally trained personnel.

Another important factor to consider in pursuit of improved diagnostictesting is the lack of, or limited availability of, freezers andrefrigeration in many third world countries. On this basis, it hasbecome more desirable to develop assay reagents that will maintain theirstability and integrity at room temperature for prolonged periods oftime. Presently, some diagnostic devices and methods require the use ofseveral assay reagents which have varying stability depending on thetemperature at which they are stored and handled. Some of these reagentsare stable at room temperature and may be stored for short periods oftime, while others are relatively unstable and begin to deterioratequickly, thereby adversely affecting the overall sensitivity andreliability of the assay. Thus, most commercially available diagnosticdevices require at least one or more of the necessary reagents be keptat low temperatures in order to ensure their stability. Accordingly, adiagnostic device incorporating reagents that can be stored at ambienttemperatures and remain stable for long periods of time while retainingall, or most, of its initial activity would have a clear advantage overcurrent state of the art devices. On this basis, a factor worthconsidering towards simplifying diagnostic testing and thus, making itmore practical and widely operational, is to minimize the number ofassay reagents (e.g. mixing, washing, diluting solvents, etc.) andintegrated steps in the assay protocol.

An immunodiagnostic assay which is simple to use, rapid and reliablewould also be advantageous in improving screening and diagnosticservices. According to the U.S. Center for Disease Control andPrevention report, rapid diagnostic tests enable healthcare providers tosupply within minutes the test results to patients at the time oftesting, thus potentially increasing the overall effectiveness ofcounseling and testing programs. It would also be expected thatsimplification of diagnostic devices and assays would likely be lesscostly to manufacture and perform compared to other conventionaldevices, thus making them economically feasible and more affordable touse in the interim. This is particularly desirable in third worldcountries where a simple, rapid, sensitive, and economical diagnosticdevice and assay would be ideal.

Towards this end, numerous analytical devices in an wide assortment ofshapes, configurations and formats have been developed for detecting thepresence of a target analyte in a fluid test sample, includingchromatographic test strips, dipsticks, lateral flow and flow-throughsystems, to name a few. Many of these devices employ reaction membranesonto which a capture reagent capable of recognizing and binding to thetarget analyte is immobilized. In essence, the method of performing theassay typically involves applying a fluid test sample suspected ofcontaining the target analyte, either directly or indirectly byfiltration, to the reaction membrane. If the target analyte is presentin the sample, it will bind to the capture reagent. Subsequent methodsare then employed to determine whether the target analyte has bound tothe capture reagent, thus indicating its presence in the sample.

U.S. Pat. No. 4,517,288 (Giegel, et al.) discloses methods forconducting ligand-binding assays using inert porous materials. Inparticular, the patent discloses immobilizing an immunological bindingmaterial (e.g. antibody) specific for the ligand of interest (e.g.antigen) within a finite test zone of the porous material and applyingthe ligand to the test zone, which will be captured by the immobilizedbinding material. Immobilization of the binding material to the porousmaterial may be achieved by any number of conventional methods includingadsorption, covalent bonding, use of a coupling agent, etc. Anenzyme-labeled indicator reagent, which will also recognize and bindwith the ligand, is then applied to the test zone where it will becomeimmobilized in an amount directly proportional to that of ligand presentin the zone. A solvent is then applied to the center of the test zone toremove any unbound indicator reagent, thus enabling the determination ofa signal to be made, with or without the aid of appropriate analyticalinstruments.

A more sophisticated version of a specific binding assay is described inU.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537 (Deutsch, et al.),which incorporates a chromatographic test strip capable of transportinga developing liquid by capillary action. The test strip is designed sothat it has a first zone for receiving a sample, a second zoneimpregnated with a first reagent capable of being transported by thedeveloping liquid and a third zone impregnated with a third reagent. Inaddition, the device comprises a measuring zone and a retarding elementwhich may be either the second reagent or the material of the strip. Thefirst reagent is capable of reacting with one of the group consisting of(1) the sample, (2) the sample and the second reagent, or (3) the secondreagent in competition with the sample, to form a product in an amountdependent on the characteristic being determined. A sample is contactedwith the first zone and the strip is then dipped into the developingliquid to bring about transport of the sample and the first reagent toform the reaction product. The retarding element slows transport ofeither the product or the first reagent (the moving reagent) tospatially separate the two and the amount of the moving element is thenmeasured at the measurement location.

A variation of the device by Deutsch, et al. is described in U.S. Pat.No. 4,960,691 (Gordon et al.) for the analysis of antigens, antibodiesor polynucleotides, which also uses a length of a chromatographicmaterial (i.e. test strip), a solvent carrier and mobile reagents.Essentially, the strip has three separate zones comprising a first zoneimpregnated with a mobile reagent reactive with the analyte of interest,a second zone for receiving a test sample suspected of containing theanalyte, and a third zone impregnated with an immobilized reagent whichselectively binds to the analyte, thereby rendering the analyte in animmobilized form. Each zone is sequentially located an equidistant fromits neighbour along a longitudinal axis of the test strip. The deviceoptionally comprises fourth and fifth zones impregnated with indicatorreagents that will provide a means of detecting the presence of theanalyte. The method involves depositing the test sample in the secondzone, followed by solvent addition to the strip at the end where thefirst zone is located so that sequential movement and arrival of theanalyte and first reagent eventually occurs at the third zone. The siterelationship between the second and third zones is such that the analyteis immobilized against solvent transport at the third zone prior to thefirst reagent reaching the third zone. Any interfering non-analytesample components, which are reactive with the first reagent, arecleared from the third zone by solvent transport prior to the arrival ofthe first reagent to the third zone. Multiple and single pathway devicesare also disclosed for accomplishing a variety of multi-step assayprocedures.

U.S. Pat. No. 4,168,146 (Grubb, et al.) discloses the use of test stripsfor carrying out sandwich-type immunoassays. The strips are formed ofbibulous carrier materials to which antibodies have been attached byadsorption, absorption or covalent bonding. Preferred test stripmaterials include cellulose fibre-containing materials such as filterpaper, ion exchange paper and chromatographic paper. Also disclosed areuses of materials such as cellulose thin-layer chromatography discs,cellulose acetate discs, starch and three-dimensional cross-linkedmaterials such as Sephadex (Pharmacia Fine Chemicals, Uppsala Sweden).The immunoassay is performed by wetting the test strip with a measuredvolume of a test sample suspected of containing the antigen. Any antigenpresent in the test sample migrates by capillary action along the teststrip. However, the extent of migration of the antigen over a fixed timeperiod is determined by the antigen concentration in the test samplebecause the bound antibodies retard the migration of the antigens forwhich they are specific. Afterwards, the antigen-containing areas of thediagnostic device are indicated by the addition of labeled antibodies.

An immunodiagnostic flow-through system comprising a series of methodsteps is disclosed in U.S. Pat. No. 4,632,901 (Valkirs, et al.). Thefirst step involves taking a fluid test sample suspected of containing afirst member of a specific binding pair (e.g. antigen) and pouring itonto a porous material to which a second member of the specific bindingpair (e.g. antibody) is immobilized. Influenced by the capillary actionproperties of an absorbent material, the fluid test sample is drawndownwards in a vertical direction through the porous material and passthe immobilized antibody. Any antigen present in the sample willsubsequently be captured by the immobilized antibody. The second stepinvolves passing a separate solution of labeled antibody through theporous material so that the labeled antibody may bind to the antigenalready captured by the immobilized antibody to form a three-memberedcomplex. Any unreacted or unbound labeled antibody is then flushed awayfrom the porous material via a third step, normally referred to as awashing step, using a suitable reagent which may then be followed by anincubation period. Finally, a fourth step involving a separate solutioncontaining a substrate reactive with the label on the antibody of thesecond solution is added to cause a visible color change indicative ofthe presence of the antigen of interest. To facilitate accurateperformance of this method, the apparatus is designed in such a way asto funnel the sample through to the absorbent material which, bycapillary action, draws the sample through the material and into thebottom of the apparatus.

An immunodiagnostic flow-through system described by Liotta in U.S. Pat.No. 4,446,232 utilizes a combination of two different reaction zonesarranged in three separate layers. The first reaction zone comprises twolayers fabricated from porous material wherein the first and secondlayers are impregnated with soluble enzyme-linked antibody andimmobilized antigen, respectively. The third layer, or second reactionzone, contains immobilized indicator reagent that will react with theenzyme linked to the antibody of the first reaction zone to produce acolor. If a liquid sample contains the antigen of interest, then afterthe sample is applied to the first reaction zone, the antigen containedtherein will bind with the soluble enzyme-linked antibody and diffusethrough to the second reaction zone following a short incubation period.The presence of antigen will be detected when the enzyme reacts with theindicator reagent to produce a color. By contrast, if a liquid sampledoes not contain any antigen, then the enzyme-linked antibody willmigrate to the second layer of the first reaction zone, aided bydiffusion of the fluid test sample, where it will bind to immobilizedantigen. The binding reaction that occurs will prevent any enzyme-linkedantibody from reaching the second reaction zone where it would reactwith the indicator reagent. Thus, in this particular scenario, no coloris observed indicating the lack of antigen in the fluid test sample.

While the methods and devices described above may provide compact andsomewhat reliable means for performing immunodiagnostic assays, severalproblems regarding their use still exist. In particular, one of thedisadvantages encountered in determining the presence or absence of atarget analyte in the majority of cases is the requirement to performseveral addition and washing steps using a range of solvents. Thewashing steps are essential at various stages of the assay protocol inorder to prevent undesired cross-reactions and to remove any excessunbound reagents and substances which may subsequently interfere withthe results. Unfortunately, this only complicates the overall procedureand effectively reduces the level of efficiency desired in order todevelop an improved and simplified version of an immunodiagnostic assay.Thus, the need to adhere to several addition, washing and incubationsteps has largely limited these procedures to clinical settings whereskilled personnel and sophisticated equipment are available to carefullymonitor and perform the assay with precision and accuracy.

In addition, immunodiagnostic assays that employ chromatographic teststrips or dipsticks suffer from a problem regarding sequential treatmentwith one or more solvents at various stages of the assay procedure. Aseach solution is added to the device, or as each device immersed intosuccessive solutions, the opportunity for spillage or contact betweenthe solutions and the user are enhanced, thus leading to possiblecontamination and reduction in the reliability of the test.

Depending on the assay and device used, it is usually necessary that thetest sample be diluted with an appropriate reagent prior to applicationso that it will diffuse more easily throughout the porous materialand/or not overwhelm the concentration of the labeled reagent. However,dilution of the test sample not only reduces the speed and ease ofperforming an assay by including an additional step and reagent, but itcan also reduce the sensitivity of an assay due to the correlation ofanalyte concentration to the detection signal generated.

A further disadvantage associated with the use of some immunodiagnosticdevices, particularly those incorporating lateral-flow techniques, isthat they characteristically require long incubation periods at variousstages of the procedure. Depending on the relative mobility of theanalyte of interest, the type of reagents and solvent used, and the siterelationship between the different reaction zones, adequate time isessential in order to allow for efficient migration of all the variouscomponents along the chromatographic solid phase material. Moreover, thelateral flow technique often contributes to a higher incidence ofinaccurate results due to the tendency of mobile reagents to accumulateat, rather than clear, the periphery of the reaction zone. As a result,these reagents will often interact at the zone and produce colorproducts that may be easily mistaken for a true positive or negativeresult.

Accordingly, the present invention provides an improved rapid diagnosticdevice, assay and multifunctional buffer for the detection of a targetanalyte in a fluid test sample which is efficient, reliable andpractical to perform. The simplified 2-step assay utilizes amultifunctional buffer reagent and a dual component flow-through devicecomprising a test unit in combination with a detachable dried indicatorreagent delivery unit which are capable of receiving the fluid testsample and multifunctional buffer, respectively.

The multifunctional buffer serves as a combination washing, diluting,wetting and resolubilizing reagent, without sacrificing the sensitivityor specificity of the diagnostic assay. Additionally, the buffer isformulated to preserve and optimize protein stability, as well asminimize, if not eliminate, non-specific interactions that might lead tothe generation of a false signal.

SUMMARY OF THE INVENTION

An object of the present invention is to overcome the disadvantagesassociated with existing rapid diagnostic assays by providing animproved device, method and multifunctional buffer reagent.

Using the simplified device and single buffer reagent of the presentinvention, a qualitative and semi-quantitative assay (1) can beperformed and read easily, (2) requires a minimum number of steps, (3)does not require lengthy incubation periods, and (4) is highlysensitive, specific and reliable. Typically, as little as a single drop(50 μL) of a fluid test sample is needed to perform the assay. Moreover,the device and assay of the present invention is particularlyadvantageous in that it is not only convenient and simple to use, butthe device and reagents can be stored at room temperature for longperiods of time without diminishing the activity or sensitivity of theassay.

The combination of features associated with the present inventionrelates to the implementation of a flow-through technique in conductingdiagnostic assays that are based on specific binding reactions betweentwo or more complementary members. On this basis, the rapid diagnosticdevice, assay and multifunctional buffer of the present invention havebroad applicability in a variety of specific binding pair assay methodsthat essentially employ a capture reagent which will recognize and bindto a target analyte of interest. For example, the device of thisinvention can be used in an immunodiagnostic assay for the detection ofeither antigen or antibody in a fluid test sample and is adaptable foruse in a sandwich or competitive detection format.

The kinetics of the reaction between the target analyte and theindicator reagent are extremely rapid and complete because the assaydevice and procedure operates on the basis of a flow-through format.Moreover, the method of the present invention improves the accuracy ofthe assay compared to conventional assays since the final step of theassay involves the addition of resolubilized indicator reagent to thetarget analyte after the analyte has already complexed with the capturereagent. By contrast, most conventional assays require premixing of theindicator reagent with a fluid test sample before addition to thecapture reagent. As a result, the overall sensitivity of the assay isreduced due to the likelihood of the indicator reagent coming intocontact with contaminants present in the test sample during the initialstage of the assay protocol, instead of only the analyte of interest.

The improved rapid diagnostic device is advantageously used incombination with a multifunctional buffer reagent for the purpose ofdetecting a target analyte in a fluid test sample based on the principleof a specific binding interaction between two or more complementarymembers. The device of the invention comprises, as a first component, atest unit capable of receiving a fluid test sample, in combination witha second component, namely a dried indicator reagent delivery unit,capable of receiving the multifunctional buffer. The test unit comprises(1) a reaction zone containing immobilized capture reagent that canspecifically recognize and bind to the target analyte, (2) an absorbentzone supporting the reaction zone, and optionally, (3) a bloodseparation zone in lateral fluid communication with the reaction zone.The dried indicator reagent delivery unit comprises a label zonepermeated with a dried indicator reagent which is resolubilized uponaddition of the multifunctional buffer. The reaction zone of the testunit is oriented so that the label zone of the dried indicator reagentdelivery unit can be brought into fluid communication therewith afterthe fluid test sample is applied to the test unit.

In the case of an immunodiagnostic assay, for example, in which theanalyte of interest is an antigen, an antibody, preferably a monoclonalantibody or an affinity purified polyclonal antibody for the antigen, isbound to the reaction zone as the capture reagent. In a preferredembodiment, the reaction zone is comprised of a porous membranecompatible for immobilization of the capture reagent and has lownon-specific binding for the indicator reagent. Any non-specific bindingsites on the surface of the porous reaction membrane are inactivated byapplying a protein blocking agent. The specificity and affinity of theimmobilized capture reagent is such that it efficiently binds andconcentrates any analyte contained in the fluid test sample within adefined region as the sample diffuses by capillary action from thereaction membrane to the absorbent zone directly underneath.

The sensitivity of reaction-membrane type immunoassays (i.e. the abilityto detect very low levels of target analyte) can be increased if thesample is concentrated through the reaction membrane. Therefore,concentration of analyte on the reaction membrane is achieved by havingan absorbent material, defining the absorbent zone, placed directlybeneath the reaction membrane that will draw the fluid test sample in,leaving only captured analyte on the upper surface of the reactionmembrane. Since the absorbent material is in fluid communication withthe reaction membrane, the material is selected on the basis of havingphysical properties (e.g. pore size, wicking power, etc.) which willeffectively induce the flow of fluid through the reaction membrane,adequately hold assay sample and reagent fluids, and provide support forthe membrane.

To facilitate the detection of a target analyte in a whole blood sample,an alternate embodiment of the present invention provides a test unitcapable of receiving and separating the fluid portion of a whole bloodsample from the red blood cells (RBC), while transporting the RBC-freefluid portion of the sample to the reaction zone for the detection ofanalyte. This particular feature is useful in preventing anyinterference during visualization of a colour reaction for the detectionof analyte (i.e. the use of “direct” labels which provide a visuallydetectable signal directly without the aid of instruments) and alsoavoids the necessity to obtain a preliminary extraction of serum orplasma in settings where proper equipment to perform such a procedure isunavailable.

Thus, in the case where the fluid test sample to be analyzed is a wholeblood sample, the test unit optionally features a separate bloodseparation zone in lateral fluid communication with the reaction zone.In general, the blood separation zone functions to selectively retaincellular components (i.e. red blood cells) contained within the wholeblood sample and deliver the remaining components of the blood sample,including any analyte, to the reaction zone. A first end of the bloodseparation zone, located a short lateral distance from the reactionzone, defines a region for receiving the whole blood sample prior tointroduction of the analyte at the reaction zone. A second end of theblood separation zone is contiguous with, and thus in direct fluidcommunication with, the reaction zone thereby promoting the capillarymovement of the RBC-free fluid portion of the blood sample from thefirst end to the reaction zone for direct analysis of the targetanalyte. Thus, in effect, the blood separation material functions as alateral flow material for the selective removal of an effective amountof red blood cells from the whole blood sample to prevent interferencewith the visual detection of the analyte, while allowing othercomponents of the sample to flow with relatively unimpaired movementthrough the test unit.

In a preferred embodiment, the blood separation zone is an elongate orrectangular strip of porous material employing a hydrophobic carrier orbacking and having intrinsic properties which enable it topreferentially entrap or retain the red blood cells in the sample withinthe blood separation zone. The carrier or backing provides support forthe blood separation material and reduces seepage of the whole bloodsample as the RBC-free fluid portion migrates along the material towardsthe reaction zone.

The second component of the device, namely the dried indicator reagentdelivery unit, comprises a label zone permeated with a dried indicatorreagent. The label zone of the dried indicator reagent delivery unit iscapable of being placed in transient fluid communication with thereaction zone of the test unit shortly following application of thefluid test sample to the test unit.

Impregnating the label zone of the dried indicator reagent delivery unitwith a permanently detectable indicator reagent eliminates the need toperform separate resolubilization steps involving precise measuring,adding and premixing with a suitable solvent which increases thepossibility of user error. In a preferred embodiment, the label zonecomprises a filter medium selected on the basis of having a pore sizelarge enough so that when the dried indicator reagent is resolubilizedby addition of the multifunctional buffer, it will easily flow throughan exposed area of the porous filter medium by the process of diffusion.The shape and dimensions of the dried indicator reagent delivery unitare such that it will hold and effectively channel the multifunctionalbuffer through the porous filter medium when the label zone is placed intransient fluid communication with the reaction zone of the test unitduring the assay procedure.

According to another important aspect of the invention, methods anddevices are provided utilizing “direct” labeled specific bindingmaterials (i.e. colloidal particle labeled materials) which are driedonto a filter medium and hence, are capable of being rapidlyresolubilized and transported to the reaction zone in the presence ofthe multifunctional buffer. Direct labels are well known in the art andhighly advantageous for their use in rapid diagnostic systems. Directlabels are capable of producing a visually detectable signal without theaid of instrumentation or the addition of ancillary reagents and arestable when stored in the dry state. Supplying the indicator reagent byway of incorporating it within the filter medium in a dried formprovides an inexpensive and convenient means of storing such reagent.The preferred label for carrying out diagnostic assays is colloidalmetal particles, more preferably colloidal gold, although other directlabels may be employed which include, but are not limited to, non-metalsols, dye sols, latex particles, carbon sol, and liposome containedcolored bodies.

According to a further important aspect of the present invention, thereis provided an aqueous composition suitable for use as a multifunctionalreagent in a diagnostic assay, comprising: (1) a biological buffer tomaintain the pH between about 7.0 to 10.0; (2) at least one surfactantto reduce non-specific binding of assay reagents while simultaneouslyavoiding inhibition of a specific binding interaction; (3) a highmolecular weight polymer as a dispersing and suspending reagent having amolecular weight in a range of from about 2×10² to about 2×10⁶ D; (4) apH stabilizer to maintain the pH of the multifunctional buffer betweenabout pH 7.0 to 10.0; (5) an ionic salt to reduce the non-specificbinding of antibodies; (6) at least one preservative to reduce bacterialand microbial growth; and (7) a calcium chelator to prevent a wholeblood test sample from clotting; wherein the biological buffer,surfactant, high molecular weight polymer, pH stabilizer, ionic salt,preservative and calcium chelator are all at effective concentrations.

The improved buffer formulation does not require ancillary additives orthe maintenance and inspection by laboratory instruments. Moreimportantly, however, is the multifunctional nature of the bufferreagent which enables it to serve as a combination wash solution,diluent, resolubilization and solvent transport reagent, therebyeliminating the need for several separate solutions and steps to beperformed during the assay protocol. The development of a singlemultifunctional buffer greatly simplifies the assay procedure byreducing the time and manual steps required to perform the assay,thereby minimizing the likelihood for user error. In addition, utilizingthe multifunctional buffer in a flow-through format promotes quickrelease and enhanced mass transfer of the dried indicator reagent fromthe dried indicator reagent delivery unit to the test unit immediatelyfollowing resolubilization. Other functional properties exhibited by themultifunctional buffer are that it maintains protein stability, therebypreserving and optimizing the specific binding reaction that occursbetween complementary binding members, i.e. capture reagent and targetanalyte. Moreover, upon resolubilization of the dried indicator reagent,the buffer helps to maximize signal generation in the case of a specificbinding reaction and minimize nonspecific binding to the reactionmembrane that might otherwise lead to the generation of a false signal.

According to yet a further aspect of the present invention, there isprovided a simple 2-step procedure for performing a diagnostic assaycomprising (1) depositing a fluid test sample onto the reaction zone ofthe test unit, or if a whole blood sample, onto a first end of a bloodseparation zone and shortly thereafter, bringing the test unit and thedried indicator reagent delivery unit into operable associationtherewith such that the label zone of the dried indicator reagentdelivery unit is in transient fluid communication with the reaction zoneof the test unit, and (2) adding the multifunctional buffer to the driedindicator reagent delivery unit followed by removal of the driedindicator reagent delivery unit to observe the test result. Followingaddition of the multifunctional buffer to the dried indicator reagentdelivery unit, the buffer reagent diffuses through the label zone toreconstitute the indicator reagent and transport it to the reaction zonewhere it will bind with any captured analyte. If analyte is present inthe fluid test sample, a detectable signal will appear in the reactionzone which can be visually inspected for color and thus, a determinationof the presence or absence of analyte made following removal of thedried indicator reagent delivery unit. An important advantage providedby the present invention is that the binding affinity of the capturereagent is capable of immobilizing and optimizing exposure of theanalyte in the flowing stream of reconstituted indicator reagent so thatit is accumulated in the reaction zone and thus, efficiently separatedfrom the background stream of non-concentrated indicator reagent.

The present invention also provides a diagnostic test kit for use in thedetection of a target analyte in a fluid test sample suspected ofcontaining the analyte. Essentially, the kit comprises in a packagedcombination: (1) the rapid diagnostic assay device comprising both thetest unit and dried indicator reagent delivery unit as described above;(2) a multifunctional buffer reagent for reconstitution of the driedindicator reagent; and (3) instructions for performing the diagnosticassay. The test kit preferably includes a suitable container for housingthe test unit and the dried indicator reagent delivery unit in order tosafeguard the solid phase materials and dried indicator reagent fromcontamination, as well as to provide ease and convenience in handling ofthe assay device. Optionally, the test kit also includes a means forapplying the test sample and multifunctional buffer to the test unit anddried indicator reagent delivery unit, respectively (e.g. disposablepipettes).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagrammatic illustration of a first embodiment of theflow-through diagnostic device of the present invention comprising thetest unit and dried indicator reagent delivery unit;

FIG. 1B is a diagrammatic illustration of a second embodiment of theflow-through diagnostic device of the present invention for analyzing awhole blood test sample comprising the test unit and dried indicatorreagent delivery unit;

FIG. 2A is a diagrammatic illustration of a test sample applied to thereaction zone of the test unit which contains target analyte;

FIG. 2B is a diagrammatic illustration of target analyte complexed withthe capture reagent after the test sample has completely diffusedthrough the reaction zone and into the absorbent zone of the test unit;

FIG. 2C is a diagrammatic illustration of the dried indicator reagentdelivery unit in fluid communication with the reaction zone of the testunit, to which the multifunctional buffer is added;

FIG. 2D is a diagrammatic illustration of resolubilized indicatorreagent reacted with complexed capture reagent and analyte followingaddition of the multifunctional buffer to the dried indicator reagentdelivery unit;

FIG. 3A is a diagrammatic illustration of a test sample applied to theporous reaction membrane of the test unit which does not contain targetanalyte;

FIG. 3B is a diagrammatic illustration of uncomplexed capture reagentafter the test sample has diffused through the reaction membrane andinto the absorbent material of the test unit;

FIG. 3C is a diagrammatic illustration of the dried indicator reagentdelivery unit in fluid communication with the reaction zone of the testunit, to which the multifunctional buffer is added;

FIG. 3D is a diagrammatic illustration of unreacted indicator reagentfollowing resolubilization by the multifunctional buffer after diffusingthrough the reaction zone and into the absorbent zone of the test unit;

FIG. 4 shows an exploded cross-sectional view of an example of asuitable container which houses the test unit and the dried indicatorreagent delivery unit;

FIG. 5 shows an enlarged cross-sectional view of the container of FIG. 4in its assembled form;

FIG. 6 is a diagrammatic illustration of a second embodiment of aportion of the test unit comprising a material defining the bloodseparation zone in fluid communication with the reaction zone; and

FIG. 7A is a diagrammatic illustration of a top plan view of the topmember of a 2-reservoir test cartridge for receiving and analyzing awhole blood sample; and

FIG. 7B is a diagrammatic illustration of a top plan view of the bottommember of a 2-reservoir test cartridge for receiving and analyzing awhole blood sample.

While this invention is satisfied by embodiments in many differentforms, there will herein be described in detail preferred embodiments ofthe invention, with the understanding that the present disclosure is tobe considered as exemplary of the principles of the invention and is notintended to limit the invention to the embodiments illustrated anddescribed. The scope of the invention will be measured by the appendedclaims and their equivalents.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. It must also be noted that, asused in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. For example, reference to an “antigen” or “antibody”is intended to include a plurality of antigen molecules or antibodies.

Ranges may be expressed herein as from “about” or “approximately” oneparticular value and/or to “about” or “approximately” another particularvalue. When such a range is expressed, another embodiment includes fromthe one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

As employed throughout the disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meanings:

Absorbent Zone—the term “absorbent zone” is intended to include one ormore layers of a permeable (e.g. porous or fibrous) material, whichlayers can be the same or different, and are capable of drawing orwicking fluid by capillary action. The absorbent zone should also becapable of absorbing a substantial volume of fluid that is equivalent toor greater than the total volume capacity of the material itself, andthus have a high absorbent capacity.

Analyte (or target analyte)—the compound or composition of interest tobe detected in a biologically derived fluid test sample. Examples ofanalytes may include drugs, hormones, polypeptides, proteins includingimmunoglobulins, polysaccharides, nucleic acids, and combinationsthereof.

Antibody—an immunoglobulin, whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteinhaving a binding domain which is, or is homologous to, an antibodybinding domain. These can be derived from natural sources, or they maybe partly or wholly synthetically produced. Examples of antibodies arethe immunoglobulin isotypes and their isotypic subclasses; fragmentswhich comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd;and diabodies.

Antibodies useful in conducting the immunoassays of the presentinvention include those specifically reactive with various analytes thedetection of which in biological fluids is desired. Such antibodies arepreferably IgG or IgM antibodies or mixtures thereof, which areessentially free of association with antibodies capable of binding withnon-analyte molecules. The antibodies may be polyclonal or monoclonaland are commercially available or may be obtained by mouse ascites,tissue culture or other techniques known to the art. A typicaldescription of hybridoma procedure for the production of monoclonalantibodies may be found in Wands, J. R., and V. R. Zurawski,Gastroenterology 80:225 (1981); Marshak-Rothstein, A., et al.; J.Immunol. 122:2491 (1979); Oi, V. Y. and L. A. Herzenberg,“Immunoglobulin Producing Hybrid”, Mishell B. B. and S. M. Shiigi (eds)Selected Methods in Cellular Immunology, San Francisco: W. H. FreemanPublishing, 1979; and U.S. Pat. No. 4,515,893 issued to Kung, et al. Theuse of mixtures of monoclonal antibodies of differing antigenicspecificities or of monoclonal antibodies and polyclonal antibodies maybe desired. It is further contemplated that fragments of antibodymolecules may be used as specific binding reagents according to theinvention including half antibody molecules and Fab, Fab′ or F(ab′)₂fragments known in the art. Regardless of the particular source or typeof antibodies, however, it is preferred that they be generally free ofimpurities. The antibodies may be purified by column chromatographic orother conventional means but are preferably purified according to knownaffinity purification techniques. Antibodies materials may also belabeled with colloidal particles according to the invention and used insandwich type assays for the detection of antigen analytes or incompetition assays for the detection of antibody analytes.

Antigen—antigens and haptens useful in carrying out the immunoassays ofthe present invention include those materials, whether natural orsynthesized, which present antigenic determinants for which the analyteantibodies are specifically reactive when used according to the presentinvention. Synthesized antigens include those which are constructedaccording to conventional chemical syntheses as well as thoseconstructed according to recombinant DNA techniques. Antigen materialsmay also be labeled with colloidal particles according to the inventionand used in sandwich type assays for the detection of antibody analytesor in competition assays for the detection of antigen analytes.

Blood Separation Zone—The term “blood separation zone” is intended toinclude a porous and/or fibrous material which is capable of retainingred blood cells (RBC) from a whole blood sample allowing the RBC-freefluid, including any target analyte, to migrate in a lateral flow by wayof capillary action.

Capilliary Action—as used herein, the term “capillary” includes acapillary or other channel or pathway which permits a liquid to traversea porous, fibrous or absorbent material. The material in capillarycommunication with the reaction membrane of the test unit is selected onthe basis of having intrinsic properties which enable it induce flow ofa fluid, either vertically or laterally, without the use of externalmeans.

Capture Reagent—any compound or composition capable of recognizing aparticular spatial and/or chemical structure of an analyte. In the caseof an analyte which is a specific immunoglobulin species, the capturereagent may be the specific protein or eptitope recognized by theimmunoglobulin. Other types of capture reagents include naturallyoccurring receptors, antibodies, antigens, enzymes, Fab fragments,lectins, nucleic acids, avidin, protein A, and the like.

Fluid test sample—the fluid test sample is assayed to form a detectiblereaction product on the reaction membrane of the test unit. In preferredassay embodiments, the fluid test sample is biologically derived (e.g.whole blood, plasma, serum, urine, saliva, etc.) and is suspected toinclude as the target analyte, typically an antigen, antibody, or haptencapable of being bound by the capture reagent immobilized on thereaction membrane.

Indicator Reagent—a conjugate comprised of a specific binding member tothe target analyte and a label conjugated to the specific binding memberwhich is capable of being visually detected. Additionally, the indicatorreagent can be comprised of a general marker protein, e.g. Protein A,Protein G, or anti-IgG conjugated to a label. For example, in an assayfor detecting antibody as a target analyte, a preferred indicatorreagent would be protein A labeled with colloidal gold. Other indicatorreagents may also include a labeled anti-human antibody directed to theantibody of interest, e.g. goat anti-human IgG labeled with colloidalgold for the detection of human antibody in a fluid test sample.

Label—a label may be any molecule bound or conjugated to a specificbinding member or general marker protein which can produce a signal. Inthe subject invention, the label is preferably a “direct” label which iscapable of spontaneously producing a detectible signal without theaddition of ancillary reagents and will be easily detected by visualmeans without the aid of instruments. The preferred embodiment of theinvention uses colloidal gold particles as the label. Other suitablelabels may include other types of colloidal metal particles, minutecolored particles, such as dye sols, and coloured latex particles. Manysuch substances will be well known to those skilled in the art.

Label Zone—The term “label zone” is intended to include a porousmaterial which is impregnated with a dried indicator reagent that can bereadily resolubilized upon addition of a buffer reagent thereto.

Reaction Zone—the term “reaction zone” is intended to include a porousmaterial to which the capture reagent and other molecules employed inthe analytical assay are bound as well as additional porous supportingmaterial, if any, that forms the lower surface of the reaction zone.

Specific Binding Member—this describes two or more complementary membersof a specific binding interaction which have binding affinity for oneanother. The specific binding members may be naturally derived orsynthetically produced. One member of the specific binding interactionhas an area on its surface, or a cavity, which specifically binds to andis therefore complementary to a particular spatial and/or chemicalstructure of the other complementary member. Examples of types ofspecific binding pairs are antigen-antibody, biotin-avidin/streptavidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate, and thelike.

1.0 INTRODUCTION

The present invention provides an improved rapid diagnostic device,assay and a multifunctional buffer for the detection of a target analytein a fluid test sample, such as a body fluid. The rapid diagnosticdevice is not only simple to use and economical to manufacture, but itis reliable enough to be utilized in sensitive analytical assays withoutrequiring lengthy incubation periods, extra washing steps, or dilutionof the sample. Since the assay may be varied according to the targetanalyte in question, the present invention is useful for a wide varietyof biological assays. For instance, a fluid test sample (e.g. serum,plasma, whole blood, saliva, urine, etc.) may be quickly and accuratelyanalyzed for antigen, antibodies, natural or synthetic steroids,hormones, and the like.

The rapid diagnostic device useful in the practice of the invention is adual component flow-through system comprising a test unit and a driedindicator reagent delivery unit capable of receiving the fluid testsample and multifunctional buffer, respectively. The test unit comprisesa reaction zone containing immobilized capture reagent that canspecifically recognize and bind to the target analyte and an absorbentzone supporting the reaction zone. The reaction zone of the test unit isoriented so that the label zone of the dried indicator reagent deliveryunit can be brought into transient fluid communication therewith shortlyafter the fluid test sample is applied to the reaction zone of the testunit. To facilitate the detection of a target analyte in a whole bloodsample, an alternate embodiment of the present invention provides a testunit further comprising a blood separation zone in lateral fluidcommunication with the reaction zone, whereby a first end of the bloodseparation zone located a short lateral distance from the reaction zonedefines a region for receiving the whole blood sample. A second end ofthe blood separation zone may overlap slightly with the reaction zone soas to ensure direct fluid communication therewith. The dried indicatorreagent delivery unit comprises a label zone containing a driedindicator reagent and is capable of being placed in transient fluidcommunication with the reaction zone of the test unit during the assayprocedure.

The assay protocol is a simple 2-step procedure involving (1) depositinga fluid test sample onto the reaction zone of the test unit, or if awhole blood sample, onto a first end of the blood separation zone andshortly thereafter, bringing the test unit and the dried indicatorreagent delivery unit into operable association such that the label zoneof the dried indicator reagent delivery unit is in transient fluidcommunication with the reaction zone of the test unit, and (2) addingthe multifunctional buffer to the dried indicator reagent delivery unitand removing the dried indicator reagent delivery unit to observe thetest result. The multifunctional buffer passively diffuses through thelabel zone of the dried indicator reagent delivery unit to resolubilizethe indicator reagent and transport it to the reaction zone of the testunit where it will bind to the corresponding analyte complexed with thecapture reagent. If analyte is present in the fluid test sample, adetectable signal will appear in the reaction zone which can be easilyvisualized following removal of the dried indicator reagent deliveryunit from the test unit. An advantage provided by the methodology of thepresent invention is the enhanced sensitivity and reliability of thetest. This is achieved by maximizing the opportunity for thoroughcapture of the analyte, even at low concentrations. Additionally, theimplementation of assay steps which increase the likelihood ofcontamination of the sample and reagents is eliminated altogether by theassay of the present invention.

2.0 SPECIFIC BINDING REACTION

The assay device of the present invention is used to qualitatively andsemi-quantitatively detect the presence of a target analyte in a fluidtest sample. Analytes suitable for detection in the assay device areessentially members of a specific binding interaction such that one ofthe members is able to recognize and bind, usually non-covalently, to acomplementary, non-identical member so as to form a stable complex thatcan be easily be detected, either directly or indirectly. The members ofthe specific binding reaction may be referred to as a target analyte anda capture reagent and may include a wide variety of biologically derivedsubstances that may participate in an immunological reaction, e.g.antigen-antibody, or a non-immunological reaction, e.g. avidin andbiotin, cell surface receptor and an effector agent, DNA and RNA, and soforth. For a disclosure of specific binding members see U.S. Pat. No.3,996,345 (Ullman, et al.).

As applied to binding assays, the assay device of the present inventioncan be designed to detect any number of target analytes, for which thereis a specific binding partner. The analyte usually is a peptide,protein, carbohydrate, glycoprotein, steroid, or other organic orinorganic molecule for which a specific binding partner exists in abiological system, or can be synthesized. The binding assay essentiallyinvolves the specific binding of the analyte (i.e. the first specificbinding member) to a capture reagent (i.e. the second specific bindingmember) immobilized on a solid phase material and additionally, anindicator reagent (comprising a label attached to an ancillary secondspecific binding member or a general marker protein). The immobilizationof the capture reagent to the solid phase material forms a “capturesitus” and thus, facilitates the separation or removal of the targetanalyte from other components of the test sample. The label, whichenables the indicator reagent to produce a detectable signal signifyingthe presence of analyte in the fluid test sample, is achieved throughdirect or indirect binding of the binding member of the indicatorreagent. Generally, the ancillary second specific binding member bindsto the target analyte at a site which does not interfere with thespecific binding interaction between the target analyte and the capturereagent. Exemplary, but not exclusive of the present invention, is thespecific binding interaction that occurs as a result of antibody-antigeninteractions.

It will be appreciated by those skilled in the art that while the rapiddiagnostic assay device described herein is anticipated to be primarilyemployed in assaying either antigens or antibodies through the formationof an immune complex, that in fact, its applicability is considerablybroader, and is not restricted to these molecules. At a minimum, thedevice merely requires that a first member that recognize and bind witha second member of a specific binding reaction. The first member can beconveniently termed a target analyte and the second member a capturereagent. While antigen and antibody are preferred embodiments of atarget analyte and capture reagent, serving respective or alternativeroles, the device can be used with a variety of capture reagent andanalyte molecules. For example, hormone receptor molecules are a type ofcapture reagent molecule and can be attached to the reaction zone of thetest unit and used to assay for the corresponding hormone analyte.Alternatively, a hormone could be bound to the reaction zone and used toassay for hormone receptors (Hermanson, G. T. (1996) BioconjugateTechniques, Academic Press).

The system is also adaptable to the detection of DNA sequences. Forexample, a fluid test sample suspected of containing a DNA sequence astarget analyte is deposited on the reaction zone and binds to a knowncomplimentary DNA sequence immobilized as capture reagent on thereaction zone. Then, a labeled DNA probe is transported by way of themultifunctional buffer to the reaction zone. If hybridization occurs,the labeled DNA probe will be retained in a visually detectable form onthe surface of the reaction zone. This system is described inPolsky-Cynkin, R., et al., Clin. Chem. 31/9, 1438 (1985).

Thus, it will be readily apparent to those skilled in the art that thereare many such combinations of capture reagent-target analyte pairs thatmay be suitably employable in the present diagnostic device and method.

3.0 ASSAY DEVICE AND METHODOLOGY 3.1 Sandwich Technique

A preferred embodiment of the present invention employs a direct binding(sandwich) assay format. The format is based on the principle of aspecific binding interaction that will occur between a target analytecomprising the first specific binding member, a capture reagentcomprising a second specific binding member that is immobilized to asolid phase material, and a dried indicator reagent comprising anancillary second specific binding member, or a general marker protein.The aforementioned members form a three-membered complex when thecontents of a fluid test sample containing the target analyte arereacted with immobilized capture reagent, followed by the addition ofthe indicator reagent. In general, the diagnostic assay thus dependsupon the ability of a second specific binding member to specificallyrecognize and bind to the first specific binding member. Depending uponthe type of target analyte to be detected, an indicator reagentcomprising an ancillary second specific binding member labeled with avisually detectable moiety is employed to determine the existence ofsuch binding. The amount of indicator reagent detected and measuredafter the reaction can be correlated to the amount of analyte present inthe test sample. For example, in the sandwich immunoassay format, a testsample containing an antigen, i.e. the target analyte, is contacted witha primary antibody which is immobilized on a solid phase material, i.e.the capture reagent. The solid phase material is subsequently treatedwith the indicator reagent, namely a secondary antibody that has beenlabeled with a visually detectable moiety. The secondary antibody thenbecomes bound to the corresponding antigen immobilized by the primaryantibody immobilized to the solid phase material and any color change isthen visually detected which is indicative of antigen present in thetest sample.

Thus, in its simplest embodiment, FIG. 1A provides a diagrammaticillustration of the assay device 1 of the present invention whichcomprises two separate components, a test unit 2 and a dried indicatorreagent delivery unit 3. The test unit 2 is comprised of a reaction zone5 having its lower surface supported by an absorbent zone 4. Thereaction zone 5 receives the fluid test sample 9 directly and providesclear visualization of a test result due to the presence of immobilizedcapture reagent 6 contained therein which is capable of recognizing andbinding the target analyte of interest through a specific bindinginteraction. In a preferred embodiment, the reaction zone 5 is comprisedof a porous membrane compatible for immobilization of the capturereagent 6 and has low non-specific binding for the indicator reagent 8.The absorbent zone 4 is preferably made from permeable materialpossessing intrinsic properties that enable it to draw fluid in bycapillary action, adequately hold reagent and sample fluids, andadditionally provide support for the reaction zone 5. The driedindicator reagent delivery unit 3 comprises a label zone 7 permeatedwith a dried indicator reagent 8. The dried indicator reagent 8comprises a label and a specific binding member which will alsorecognize and bind to the analyte of interest, but at a site which doesnot interfere with the specific binding interaction between the targetanalyte and the capture reagent. The label zone 7 preferably comprises afilter medium selected on the basis of having a pore size large enoughso that when the dried indicator reagent 8 is resolubilized by additionof the multifunctional buffer 12, it will easily flow through an exposedarea of the label zone 7 by the process of diffusion. The shape anddimensions of the dried indicator reagent delivery unit 3 are such thatit will hold and effectively channel the multifunctional buffer throughthe label zone 7 when placed in transient fluid communication with thereaction zone 5 of the test unit 2 during the final step of the assayprocedure.

FIG. 1B provides a diagrammatic illustration of the assay device 1 of asecond embodiment of the present invention which comprises two separatecomponents, a test unit 2 and a dried indicator reagent delivery unit 3,wherein the test unit 2 additionally comprises a blood separation zone100 capable of receiving and separating the fluid portion of a wholeblood sample 9′ from the red blood cells (RBC), while transporting aRBC-free fluid portion, including any analyte, to the reaction zone 5for direct analysis. The preferred material for the blood separationzone 100 is selected on the basis of having intrinsic properties whichenable it to preferentially entrap or retain the red blood cells in thesample 9′ as the fluid portion migrates in a lateral direction towardsthe reaction zone 5.

FIG. 2 is a diagrammatic illustration showing the method of theinvention using the device of FIG. 1A. In this particular instance, FIG.2A shows a fluid test sample 9 containing the target analyte 10, as wellas other non-essential components 11, which is applied to the reactionzone 5 of the test unit 2. As the fluid test sample 9 diffuses throughthe reaction zone 5 and into the absorbent zone 4 underneath, the freeanalyte 10 comes into contact with available sites of attachment on thecapture reagent 6 and forms a complex, while unbound non-essentialcomponent 11 continues to be drawn into the absorbent zone 4 below (FIG.2B). As shown in FIG. 2C, the label zone 7 of the dried indicatorreagent delivery unit 3 is subsequently brought into fluid communicationwith the reaction zone 5 of the test unit 2 prior to the addition of themultifunctional buffer 12. Immediately following resolubilization of thedried indicator reagent 8 by the buffer 12, the indicator reagent 8 istransported to the reaction zone 5 of the test unit 2, where it willbind with any analyte 10 that has complexed with the capture reagent 6.The binding reaction of the indicator reagent 8 with the analyte 10produces a visually detectable signal thereby indicating a positiveresult that is easily observed following removal of the dried indicatorreagent delivery unit 3, as per FIG. 2D.

FIG. 3A is a diagrammatic illustration showing the method of theinvention using the device of FIG. 1A when a fluid test sample 9 devoidof target analyte is applied to the reaction zone 5 of the test unit 2.As the fluid test sample 9 diffuses through the reaction zone 5 and intothe absorbent zone 4 underneath, the non-essential components 11completely bypass the capture reagent 6, leaving the sites of attachmentunoccupied (FIG. 3B). As shown in FIG. 3C, the label zone 7 of the driedindicator reagent delivery unit 3 is subsequently brought into fluidcommunication with the reaction zone 5 of the test unit 2 prior to theaddition of the multifunctional buffer 12. Immediately followingresolubilization of the dried indicator reagent 8 by the buffer 12, theindicator reagent 8 is transported to the test unit 2, where it diffusesthrough the reaction zone 5, pass the capture reagent 6 and into theabsorbent zone 4 below due to the absence of any target analytecomplexed with capture reagent 6. Following removal of the driedindicator reagent delivery unit 3, a color signal will not be detectedthereby indicating a negative result due to the absence of bindingbetween the indicator reagent 8 and complexed analyte.

To facilitate the detection of a target analyte in a whole blood sample,an alternate embodiment of the present invention provides a test unithaving a blood separation zone capable of receiving and separating thefluid portion of a whole blood sample from the red blood cells (RBC),while transporting the RBC-free fluid portion, including any analyte, tothe reaction zone for direct analysis. As shown in FIG. 6, the bloodseparation zone 100 is preferably an elongate strip of porous materialwhich is selected on the basis of having intrinsic properties whichenable it to preferentially entrap or retain the red blood cells in thesample 9′ as the fluid portion migrates in a lateral direction towardsthe reaction zone 5. Although the shape and dimensions are not critical,preferably the blood separation zone 100 is a rectangular form havingdimensions suitable for allowing efficient removal of a substantialamount of red blood cells from the whole blood sample 9′ prior to theRBC-free fluid portion of the sample 9′ arriving at the reaction zone 5.Thus, in effect, the blood separation material functions as a lateralflow material for the selective removal of an effective amount of redblood cells from the whole blood sample 9′ so as to avoid interferencewith the visual detection of the analyte, while allowing othercomponents of the sample, including any analyte, to flow with relativelyunimpaired movement to the reaction zone 5. Preferably, a hydrophobiccarrier 103 is affixed to the lower surface of the blood separation zone100 to provide support and reduce seepage of the fluid phase while theRBC-free fluid portion of the whole blood sample migrates towards thereaction zone 5. The carrier 103 is preferably similar in shape and sizeto the blood separation zone 100.

A first end 101 of the blood separation zone 100, located a shortlateral distance from the reaction zone 5, defines a region forreceiving the whole blood sample 9′ prior to introduction of the analyteat the reaction zone 5. A second end 102 of the blood separation zone100 is contiguous with and may overlap slightly with the reaction zone5, so as to be in direct fluid communication with the reaction zone 5,thereby promoting the capillary movement of the RBC-free fluid portionof the blood sample 9′ from the first end of the blood separation zone100 to the reaction zone 5. The blood separation zone 100 and thereaction zone 5 must contact one another in order to ensure optimaltransfer of the sample from one zone to the other. Therefore, it ispreferably that the blood separation zone 100 and the reaction zone 5overlap with one another slightly as opposed to being abutted to oneanother.

Thus, the two-step assay protocol optionally employs a simultaneousseparation of red blood cells from a whole blood sample 9′ in order topermit testing for a desired analyte without the requirement foradditional steps. For example, in the case where a whole blood sample 9′contains analyte, the sample 9′ is simply applied to the first end ofthe blood separation zone 100 of the test unit, rather than the reactionzone 5. As the RBC-free fluid portion of the blood sample 9′ migrates ina lateral direction to arrive at the reaction zone 5, the free analyteeventually comes into contact with available sites of attachment on thecapture reagent and forms a complex. Thus, similar to the method stepsshown in FIG. 2, unbound non-essential components are drawn into theabsorbent zone located beneath the reaction zone. The label zone of thedried indicator reagent delivery unit is subsequently brought into fluidcommunication with the reaction zone of the test unit prior to theaddition of the multifunctional buffer. Immediately followingresolubilization of the dried indicator reagent by the buffer, theindicator reagent is transported to the reaction zone of the test unit,where it will bind with any target analyte that has complexed with thecapture reagent. The binding reaction of the indicator reagent with thetarget analyte produces a visually detectable signal thereby indicatinga positive result that is easily observed following removal of the driedindicator reagent delivery unit.

3.2 Competitive Technique

Those skilled in the art can deduce the application of the presentinvention in competitive, as well as noncompetitive (e.g. sandwich),assays for target analyte of suitable interest. In the competitiveformat, it is an ancillary first specific binding member of theindicator reagent (as opposed to an ancillary second specific bindingmember in the case of the “sandwich” technique) which is capable ofbinding to the second specific binding member, i.e. the capture reagent.In other words, ancillary first specific binding member of the indicatorreagent competes with the target analyte, i.e. first specific bindingmember, for binding to sites of attachment of the capture reagent. Theancillary first specific binding member will comprise, for example, ananalogue or other authentic sample of the target analyte which hascomparable binding affinity with the first binding member. When thefluid test sample is deposited on the reaction zone, any target analyte,if present, will bind to available sites of attachment of the capturereagent, i.e. the second binding member, and thus potentially block theancillary first binding member of the indicator reagent from binding tothe capture reagent following its addition. If the fluid test samplehappens to contain the target analyte, the absence of a color signalwill indicate a positive result due to the inability of the indicatorreagent to bind to the capture reagent. Alternatively, if the testsample does not contain any target analyte, the presence of a colorsignal will indicate a negative result due to the ability of theindicator reagent to bind to unoccupied sites of attachment of thecapture reagent.

4.0 TEST UNIT

As described above, the diagnostic device of the present inventioncomprises, as a first component, a test unit having a reaction zonecontaining immobilized capture reagent that can specifically recognizeand bind to the target analyte, an absorbent zone supporting thereaction zone, and optionally, a blood separation zone in lateral fluidcommunication with the reaction zone. The reaction zone of the test unitis oriented so that the label zone of the dried indicator reagentdelivery unit can be brought into transient fluid communicationtherewith shortly after the fluid test sample is applied to the testunit.

4.1 Reaction Zone

The term “reaction zone” is intended to include the porous material towhich the capture reagent and other molecules employed in the analyticalassay are bound as well as additional porous supporting material, ifany, that forms the lower surface of the reaction zone.

The selection of the material for the reaction zone is not critical tothe invention. The materials used to fabricate the device of the presentinvention are well known in the art. Porous materials, such as thosedescribed in U.S. Pat. Nos. 4,670,381, 4,632,901, 4,666,863, 4,459,361,4,517,288, and 4,552,839, may be composed singly or in combination ofglass fibers, cellulose acetates, nylon, or various synthetic or naturalmaterials.

The preferred material of the reaction zone is a membrane which has apore size permitting separation and filtration of other non-essentialcomponents from the fluid test sample being assayed. The flow of theaqueous reagents is controlled through diffusion and the membrane shouldhave low nonspecific binding for the indicator reagent before or aftertreatment with reagents such as proteins, detergents, or salts. Thereare many porous membrane, films, or papers available commercially whichhave controlled hydrophobicity and are suitable for the practice of theinvention. The reaction membrane can be any shape and thickness butusually is flat and thin. The absorption, diffusion or filtration of theliquid phase of the reactants from the solid phase particles in theseparation step of the assay can be facilitated by the addition of afibrous or hydrophilic material (absorbent pad) in contact with theunderside of the reaction membrane. The size of the area exposed to thesolid phase particles can be controlled by using a hydrophobic materialsuch as plastic, plastic laminate, or other similar substance that isplaced in contact with the reaction membrane and seals the reaction zonesuch that only a surface area no greater than about 150 mm² is exposedto the particulate solid phase.

The porosity of the membrane has a large influence on the flow rate ofthe liquid and sensitivity of the assay. The larger the pore size of themembrane, the faster the flow rate for a given liquid. As the flow rateincreases, the interaction time available between the target molecule inthe sample and the receptor immobilized on the reaction membranedecreases, thus decreasing assay sensitivity. Additionally, larger poresizes provide less surface area for immobilizing the receptor molecule,which is another parameter attributable to decreased assay sensitivity.For most assays, the porosity of the membrane is preferably in the rangeof about 0.1 to about 12.0 microns, and more preferably ranging fromabout 0.2 to 0.8 microns.

The wicking power of the membrane may also affect assay sensitivity anddepends on the thickness and nature of the membrane material. Wickingpower can be measured as the migration of a standard solution through acertain distance per unit time. Often times, selecting a membrane havinga relatively low wicking power can increase assay sensitivity. Thus, inaddition to porosity, the overall thickness of the reaction membrane mayaffect assay sensitivity and therefore, must also be considered.

The thickness of the reaction membrane, which is the distance betweenthe upper and lower surfaces of the reaction membrane, can varydepending upon the flow characteristics needed for a given diagnosticassay. Typically, the thickness will range from about 0.05 mm to aboutto 3.0 mm, and more commonly from about 0.1 to about 1.0 mm. With someimmunoassays in particular, it has been found that when the thickness ofthe reaction membrane is greater than about 0.1 mm, and preferably inthe range of about 0.2 mm to about 1.0 mm, higher sensitivity can beachieved. Moreover, it is believed that prior art devices which haverelatively thin reaction membranes, such as nitrocellulose membranesless than 0.1 mm thick and which are not paper-backed, tend to allow thesample to flow sideways across the reaction membrane rather thandownwards through the middle of the reaction membrane. On the otherhand, a thicker reaction membrane may allow more capture reagent to beavailable for binding to the target analyte, thereby providing a furtherincrease in assay sensitivity. Thus, the thickness of the membraneshould be selected so that an adequate amount of binding reagent can beimmobilized to capture the sample component. However, if the membranethickness is to large, it may cause undue delay of the passage of thefluid test sample through the membrane.

Another factor to be considered is that the material of the reactionmembrane be selected on the basis that it is compatible forimmobilization of the capture reagent. The reaction membrane may be anysuitable porous material capable of immobilizing the capture reagentemployed in the diagnostic assay so long as the performance of the assayis not adversely affected. Suitable materials include nitrocellulose(supported or unsupported), glass fiber, polyester, cellulose nitrate,polyester, polycarbon, nylon, and other natural and synthetic materialswhich can be coupled directly or indirectly to the selected capturereagent. Usually the membrane will comprise negative charges that allowthe capture reagent molecule to bind. Certain membrane materials whichare charged include cellulose nitrate which has partial negative chargescontributed by the nitro groups.

In some cases commercial filters are available that have immobilized totheir internal and/or external surfaces a reactant for the attachment ofbiological molecules, such as antibodies or antigens, to the surfaces.Examples of various filters include cellulosic filters (filter papers),polyamide membranes (e.g. numerous variations of polyamide membranes aremanufactured by the Pall Corporation), and various other microporousmembranes, such as those available commercially from Amicon, Geleman,and Schleicher & Schuell. For example, the following membranes areavailable from Pall Corporation: Biodyne®, a N66 polyamide microporousmembrane (U.S. Pat. No. 4,340,479 issued to Pall); Carboxydyne®, ahydrophilic, microporous, skinless nylon 66 membrane with controlsurface properties characterized by carboxyl functional groups at itssurfaces; and Immunodyne™, a modified Carboxydyne® membrane prepared bytreating a Carboxydyne® membrane with trichloro-s-triazine. Othermicroporous membranes, prepared by the Millipore Corporation, aredescribed in U.S. Pat. Nos. 4,066,512 and 4,246,339.

Other materials may be pre-treated to provide a charged membrane. Forexample, polyester can be derivatized with carboxyl or amino groups toprovide either a negatively or positively charged membrane. Nylon can betreated with acid to break peptide bonds to provide positive charges(from the amine groups) and negative charges (from the carboxyl groups).

A preferred material for utilization as a reaction membrane is anitrocellulose membrane backed with porous paper similar to filterpaper, or other types of nitrocellulose membranes with similarcharacteristics. A representative example is commercially availableunder the trade name BAC-T-KOTE by Schleicher and Schuell. This materialis substantially more durable than nitrocellulose alone and can beemployed without any other support component while allowing for easierhandling and device assembly. Additionally, it has been found thatanalytical devices employing paper-backed nitrocellulose for thereaction zone have enhanced sensitivity in certain immunoassays.

Other commercially available materials are from EY Laboratories Inc.(San Mateo, Calif.; Cat. Nos. PBNC15-1, PBNC15-10, PBNC15M-1, andPBNC15M-10).

4.2 Immobilization of the Capture Reagent

In a typical system, the capture reagent is immobilized on the porousmembrane of the reaction zone which will specifically recognize and bindto any target analyte present in the fluid test sample being assay. Suchreagent, typically an immunological protein such as an antibody orantigen, can be immobilized directly or indirectly onto such materials,such as nitrocellulose, by either absorption, adsorption, or covalentbonding. When a fluid test sample suspected of containing the targetanalyte of the specific binding interaction is applied to the reactionzone containing the immobilized capture reagent, it becomesnon-diffusively bound to the reaction zone. Thus, by appropriateapplication of a fluid test sample suspected of containing the targetanalyte of interest, a high concentration of the target analyte can beobtained in a well defined region within the center of the reactionzone. In appropriate cases, the capture reagent may be coated on theupper surface of the reaction zone or be a particulate which isentrapped within the matrix of the porous material of the reaction zone.Therefore, as used herein, the term “immobilized” is intended to embraceany means for fixing the capture reagent to the porous material.

A first step of the present method is to immobilize the capture reagentwithin a finite zone of the reaction zone. Immobilization can beaccomplished by methods such as adsorption, absorption, evaporativedeposition from a volatile solvent solution, covalent bonding betweenthe capture reagent and the reaction membrane, or immunologicalimmobilization. Covalent bonding may, for example, involve bonding thecapture reagent to the reaction zone through a coupling agent, such as acyanogen halide, e.g. cyanogen bromide or by the use of gluteraldehyde,as described by Grubb, et al. in U.S. Pat. No. 4,186,146. Immunlogicalimmobilization to the reaction membrane may be by absorption, or bycovalent linkage, directly, or through a linker of sorts well-known tothose skilled in the art. Suitable methods of carrying out theseprocedures are given, for example, by Iman and Hornby in BiochemicalJournal (Volume 129; Page 255; Campbell, Hornby, and Morris in Biochem.Biophys. Acta (1975), Volume 384; Page 307; and Mattisson and Nilsson inF.E.B.S. letters, (1977) Volume 104, Page 78. See also, for example,U.S. Pat. Nos. 4,376,110 and 4,452,901. In addition, chemicallypretreated materials suitable for coupling antibodies can be purchasedcommercially.

Immunological immobilization is preferred for the practice of thepresent invention. For example, if a sandwich immunoassay is employed inthe present device using antibody as the capture reagent, then thereaction membrane is impregnated with antibody by way of absorptionusing a dispenser/printer technique (BioDot, Calif., U.S.A.). Thisinvolves applying one or more distinct antibodies to the membrane byspraying them directly onto a reaction membrane. The above technique ismost readily achieved using a commercial printing device termed a BIOJETQUANTI 3000, and provides a stream of the immunological protein under avariety of conditions, and at varying stream widths. Using thistechnique, it is possible to rapidly deposit a series of lines, or otherdiscrete patterns on the reaction membrane, each containing an antibodywith different antigenic specificities for binding one or more antigens.Thus, the number of antigens that can be assayed is a function of thenumber of different antibodies that can be applied in distinct patterns.

Depending on the detection limits the user wishes to impose on thediagnostic assay, the capture reagent can be deposited singly or invarious combinations in the reaction zone in a variety of configurationsto produce different detection or measurement formats. For example, apanel of two or more different specific binding members selected as thecapture reagent for the diagnostic assay may be applied to differentregions of the same reaction membrane so that the presence of multipleanalytes in a single fluid test sample may be simultaneously analyzed,e.g. for the detection of HIV and HCV. Preferably, the capture reagentis deposited in a discrete test zone having an area substantiallysmaller than that of the entire surface area of the porous material usedin the reaction zone. Various patterns that are convenient for thedistribution of the capture reagent may include, but are not limited to,numerals, letters, dots, lines and symbols, or the like, which displaythe detectable signal upon completion of the assay. It is preferred thatthe pattern of the discrete test zone be in the form of a single line toenhance the visability of the test result.

4.3 Capture Reagent

Since the present apparatus is designed to be used in a method fordetecting a target analyte in a fluid test sample, a capture reagentmust be provided which will recognize and be capable of specificallybinding to the target analyte. One of ordinary skill in the art willappreciate that the term “specific binding” refers to the interactionthat will occur between two or more complementary non-identicalcomponents to form a complex. Examples of such binding pairs includeantigens and antibodies, hormones (and other intracellular messengers)and cell receptors, sugars and lectins. Either member of the specificbinding pair can be immobilized to the reaction zone with the othermember being the analyte being detected in the test sample. Exemplary,but not exclusive of the present invention, is the specific bindinginteraction that occurs as a result of antibody-antigen interactions.However, it should be realized that the use of terms such as antigen andantibody are not mutually exclusive since antibodies can act as antigensfor other antibodies.

Because of the relative ease with which specific antibodies can now beprepared against antigens, preferred embodiments of the invention may orcan use monoclonal antibodies attached to the reaction membrane todetect the presence of their specific antigen in a fluid test sample.The monoclonal antibodies can belong to any of the classes or subclassesof antibodies, including IgA, IgD, IgE, IgG (subclasses 1-4, if human;1, 2a, 2b, 3, if murine), or IgM. Actively binding fragments ofantibodies can also be employed, such as Fab, Fv, F(ab′)₂, or the like.The preparation of monoclonal antibodies is well known in the art whichis accomplished by fusing spleen cells from a host sensitized to theantigen with myeloma cells in accordance with known techniques or bytransforming the spleen cells with an appropriate transforming vector toimmortalize the cells. The cells can be cultured in a selective medium,cloned, and screened to select monoclonal antibodies that bind thedesignated antigens. Numerous references can be found on the preparationof monoclonal and polyclonal antibodies (e.g. Kohler and Milstein,(1975) Nature (London) 256, 495-497; Kennet, R., (1980) in MonoclonalAntibodies (Kennet et al., Eds. pp. 365-367, Plenum Press, N.Y.).

4.4 Control Zone

In addition to the capture reagent, a defined area of the exposedreaction zone may also contain a control molecule. In this regard, colordevelopment at the test site may be compared with the color of one ormore standards controls to determine whether the reagents are stable andthe test is performing properly. In general, when testing for thepresence of target analyte, the diagnostic device will have a built-incontrol of an antibody directed to human immunoglobulin G (IgG), IgM,IgE, or IgA. Thus when a fluid test sample is added to the diagnosticdevice, immunoglobulin will bind to the control region regardless ofwhether or not target analyte happens to be present in the sample. Forexample, a suitable control may be established by using Protein A whichis disclosed in U.S. Pat. No. 5,541,059 (Chu). Other suitable controlsare well known in the art.

4.5 Blocking the Reaction Zone

As noted above, the capture reagent, and the optional use of controls,are typically applied only to defined regions of the exposed surface ofthe reaction zone. The capture reagent will often be applied to a regionwithin the center of the reaction zone such that the perimeter of theexposed surface of the reaction zone will not have any capture reagentbound thereto. On the other hand, in some situations, it may beappropriate to cover the entire exposed surface of the reaction zonewith the capture reagent. If, however, capture reagent is immobilizedonto a limited region of the exposed surface of the reaction zone, theporous material or membrane from which the zone is made can be treatedwith a blocking composition that prevents the target analyte and othercomponents of the sample from non-specifically binding to the reactionzone. For assays where non-specific binding is not problematic, ablocking step will be unnecessary. Also, the use of a good qualitypaper-backed nitrocellulose may make a blocking step unnecessary in someassays. However, if a blocking step is needed, common blocking solutionscomprising bovine serum albumin (BSA) or other proteins which do notinterfere with, or cross-react with, reagent materials of the assay canbe used. BSA is usually used in amounts from about 1 to 10%.

The blocking treatment typically occurs after the analytical device hasalready been assembled and the capture reagent is immobilized to thereaction zone. A sufficient amount of blocking composition which willcover the exposed surface of the reaction zone is applied. After theblocking composition has dried, the analytical device is ready for use.

4.6 Absorbent Zone

The sensitivity of reaction-membrane type immunoassays (i.e. the abilityto detect very low levels of target substance) can be increased if thesample is concentrated through the reaction zone. With some devices,concentration of the sample through a reaction zone is achieved byhaving an absorbent material, or pad, beneath the reaction zone thatdraws the sample, which is added to the surface of the reaction zone,through to the absorbent material below. The absorbent zone can begenerated from any material capable of wicking fluid by way of capillaryaction, such as cotton or paper. Membrane-based immunoassays thatutilize various absorbent materials to concentrate sample areexemplified in U.S. Pat. Nos. 5,185,127, 5,006,464, 4,818,677,4,632,901, and 3,888,629.

An absorbent material is situated underneath the lower surface of thereaction zone so as to be in direct fluid communication with thereaction zone. Thus, the upper surface of the absorbent material isimmediately adjacent to the lower surface of the reaction zone. Fluidcommunication contact involving direct physical contact of the absorbentmaterial with the reaction zone may optionally include the separation ofa portion of the absorbent material from the reaction zone by anintervening spacer layer which has an opening therein. Accordingly, thespacer layer still permits direct contact between the reaction zone andthe absorbent zone thereby enabling the assay reagents to flow uniformlyfrom the upper surface down to the lower surface of the assay apparatus.Although not critical to the performance of the apparatus, the spacerlayer also serves to hold the porous membrane of the reaction zone. Thespacer layer may be made of any rigid or semi-rigid material that doesnot bind or interact with assay reagents used in conjunction with theinvention. Exemplary of materials for the spacer layer 25 arefiberglass, paper, hydrophilic polypropylene, or cellulose. Thethickness of the spacer layer 26 will generally be in the range of about0.1 mm to 1 mm. In embodiments of the invention where ease ofmanufacture and reduced costs are desired, the upper surface of theabsorbent material is typically placed immediately adjacent the lowersurface of the reaction zone.

The selection of material for the absorbent zone is not critical and avariety of fibrous filter materials can be used, including one or morelayers of the same or different materials, providing that the materialselected is compatible with the target analyte and the assay reagents.Any conventionally employed absorbent material that is capable ofdrawing or wicking fluid through a porous membrane, such as for example,by capillary action, can be used in the present invention. The absorbentmaterial should be capable of absorbing a volume of fluid test samplethat is equivalent or greater than the total volume capacity of thematerial itself. Useful known materials include cellulose acetatefibers, polyester, polyolefin or other such materials. The absorbentmaterial provides a means to collect the sample by providing uniform“suction” to deliver the sample from the well, through the reactionzone, and down into the absorbent material. Thus, the absorbent bodyalso acts as a reservoir to hold the sample, and various reagents thatare used when the assay is performed. Accordingly, when used in assayswhere relatively large volumes of fluid are used, the absorbent materialshould have high absorbent capacity so as to prevent or minimize thepossibility of back-flow of sample and reagents from the absorbent bodyback into the reaction membrane.

As with the reaction zone material, the wicking power of the absorbentmaterial can be an important parameter. Wicking time is defined in termsof the time required for water to travel a defined distance through theabsorbent material and is related to the thickness and porosity of thematerial. Wicking power can vary greatly from one material to the nextand therefore, the properties of the analytical device and flow rate ofsample and reagents can be modified by varying the absorbent materialused.

4.7 Blood Separation Zone

To facilitate the detection of a target analyte in a whole blood sample,an alternate embodiment of the present invention provides a test unitcapable of receiving and separating the fluid portion of a whole bloodsample from the red blood cells (RBC) featuring a blood separation zonein lateral fluid communication with the reaction zone. The bloodseparation zone functions to selectively retain cellular components(i.e. red blood cells) contained within the whole blood sample anddeliver the remaining components of the RBC-free fluid portion of theblood sample, including any analyte, to the reaction zone for eventualanalysis. This particular feature is useful in preventing anyinterference during visualization of a color reaction for the detectionof analyte and avoids the necessity of obtaining a preliminaryextraction of serum or plasma in settings where proper equipment toperform such a procedure is unavailable.

Various methods for the separation of blood cells from the fluid portionof blood are described using separation coatings, erythrocyteaggregating and agglutinating agents, materials having asymmetric poresizes, polymer-containing matrixes, and multilayer systems, to name afew, e.g. U.S. Pat. No. 3,768,978 to Grubb et al., U.S. Pat. No.3,902,964 to Greenspan, U.S. Pat. No. 4,477,575 to Vogel et al., U.S.Pat. No. 4,594,372 to Zuk, U.S. Pat. No. 4,753,776 to Hillman et al.,U.S. Pat. No. 4,816,224 to Vogel et al., U.S. Pat. No. 4,933,092 toAunet et al., U.S. Pat. No. 5,055,195 to Trasch et al., U.S. Pat. No.5,064,541 to Jeng et al., U.S. Pat. No. 5,076,925 to Roesink et al.,U.S. Pat. No. 5,118,428 to Sand et al., U.S. Pat. No. 5,118,472 toTanaka et al., U.S. Pat. No. 5,130,258 to Makino et al., U.S. Pat. No.5,135,719 to Hillman et al., U.S. Pat. No. 5,209,904 to Forney et al.,U.S. Pat. No. 5,212,060 to Maddox et al., U.S. Pat. No. 5,240,862 toKoenhen et al., U.S. Pat. No. 5,262,067 to Wilk et al., U.S. Pat. No.5,306,623 to Kiser et al., U.S. Pat. No. 5,364,533 to Ogura et al., andU.S. Pat. No. 5,397,479 to Kass et al.

In a preferred embodiment, the blood separation zone is an elongate orrectangular strip of porous material having intrinsic physicalproperties which enable it to preferentially and sufficiently entrap orretain the red blood cells in the sample within the blood separationzone. A first end of the blood separation zone, located a short lateraldistance from the reaction zone, defines a region for receiving thewhole blood sample during the first step of the assay protocol, andprior to introduction of the target analyte at the reaction zone. Asecond end of the blood separation zone, in direct fluid communicationwith the reaction zone, helps to promote the movement of the RBC-freefluid portion of the blood sample from the first end of the bloodseparation zone to the reaction zone for eventual analysis. The bloodseparation zone and the reaction zone must contact one another in orderto ensure optimal transfer of the sample from one zone to the other.Accordingly, the materials selected for the blood separation zone andthe reaction zone may overlap slightly with one another in order toensure adequate migration of the RBC-free portion of the whole bloodsample.

A variety of materials can be used for the blood separation zone such asglass fiber, glass fiber/cellulose mixtures, cellulose, or otherproprietary materials, including synthetic materials, e.g., nylon.Preferably, a permeable glass fiber matrix is employed as the bloodseparation material to facilitate the separation of red blood cells fromwhole blood. A variety of grades of different thicknesses andabsorbencies of glass fiber materials are commercially available tofacilitate blood separation and include, for example, GF-24, GF-25, and#33, available from Schleicher & Schuell (Keene, N.H., U.S.A.); G143,G144, and G167, available from Ahistrom (Mount Holly Springs, Pa.,U.S.A.); GFQA30VA, GF/P 30, GF/DE 30, GF/SE 30, GF/CM30VA, GF/CM 30, F075-14, F487-09, GF DVA, GFVA 20, and GD-2, available from Whatman(Fairfield, N.J., U.S.A.).

Useful glass fiber/cellulose mixture materials include F255-07 90glass/10 cellulose, F255-09 70 glass/30 cellulose, F255-11 50 glass/50cellulose, and F255-12 50 glass/50 cellulose, available from Whatman.

Useful cellulose materials include 598, available from Schleicher &Schuell. Miscellaneous or other materials falling outside the abovecategories can also be used, including HemaSep V and Leukosorb; whicharticle of manufacture according to the subject invention available fromPall BioSupport (Port Washington, N.Y., U.S.A.).

One useful nylon material is Nylon 6.6 Transfer Membrane, which iscommercially available under the tradename Biodyne B (Pall SpecialtyMaterials, Port Washington, N.Y.). In addition, the material known as“PlasmaSep”, available from Whatman, can be used.

Although the shape and dimensions of the blood separation zone are notcritical, preferably it has a narrow rectangular form and dimensionssuitable for allowing efficient removal of a substantial amount of redblood cells from the whole blood sample during migration of the fluidportion of the sample from the first end to the second end of the zone.Thus, in effect, while a narrow rectangular shape is preferred tochannel fluid portion of the blood sample to the reaction zone, thedimensions may vary depending on the intrinsic properties (e.g.absorbency, migration rate, etc.) of the material selected for the bloodseparation zone. In a preferred embodiment, the blood separation zone ismade using the glass fiber material F487-09, available from Whatman,having dimensions between approximately 4 to 7 mm in width, betweenapproximately 10 and 15 mm in length, and between approximately 0.2 mmand 1.0 mm in thickness. More preferably, the blood separation materialis about 7 mm in width by about 10 mm in length and about 0.5 mm inthickness. These dimensions are optimized to be capable of receiving andseparating the total volume of a whole blood sample, e.g. two drops ofblood.

The blood separation material preferably has a rigid or semi-rigidcarrier or backing affixed to its lower surface to provide support andreduce seepage of the RBC-free fluid portion of the whole blood samplewhile it migrates towards the reaction zone. Suitable materials for useas a carrier or backing include, for example, hydrophobic materials suchas polycarbonate, polyethylene, Mylar, polypropylene, vinyl, cellophaneand polystyrene, etc. as well as water-proofed or fluid-resistantcardboard or similar materials. The carrier or backing may be affixedeither directly or indirectly to the blood separation material by meansof a fluid-resistant adhesive.

Suitable adhesives are well-known in the art. The carrier may be of anyshape and of almost any size which may conveniently be handled. However,the carrier is preferably similar in shape and size to the bloodseparation material. Thus, the carrier is preferably formed as anelongate or rectangular strip having a length and width similar to orthe same as the blood separation material.

5.0 DRIED INDICATOR REAGENT DELIVERY UNIT

As discussed above, the diagnostic device of the present inventioncomprises, as a second member, a dried indicator reagent delivery unitcomprising a label zone permeated with a dried indicator reagent.

The selection of the material for the label zone is not critical and canbe any suitably absorbent, porous or capillary possessing materialthrough which the multifunctional buffer and resolubilized indicatorreagent may be transported by wicking action. The criteria of selectionis that the material allow for the resolubilization and mixing of thedried indicator reagent upon addition of the multifunction buffer, aswell as initiate the transfer of the buffer and freshly dissolvedindicator reagent to the reaction zone of the test unit.

Natural, synthetic, or naturally occurring materials that aresynthetically modified, can be used as a filter medium including, butnot limited to cellulose materials such as paper, cellulose, andcellulose derivatives such as cellulose acetate and nitrocellulose,fiberglass, cloth, films of polyvinyl chloride, and the like. Although apreferred filter medium is nitrocellulose, the material should be chosenfor its ability to release the indicator reagent upon reconstitutingwith the multifunctional buffer. Moreover, the fluid flow through thefilter medium should be laminar as opposed to turbulent flowcharacteristics which adequately allows for initial mixing of the bufferwith the indicator reagent.

5.1 Indicator Reagent

The use of indicator reagents to detect the presence of a target analytein a test sample is well known in the art. Depending on the type ofdiagnostic assay employed, the label employed in the indicator reagentis conjugated to a specific binding member or general marker protein(e.g. Protein A, Protein G, anti-IgG) that will directly, or indirectly,bind to the target analyte. Formation of an indicator reagent between aspecific binding member and a label may be any of the conventional typesincluding metal complex labels, radioactive labels, enzyme labels,fluorescent labels, radioactive labels, chemiluminesct labels, and thelike.

An important consideration in the design of a rapid diagnostic device isthat the label chosen in the generation of the indicator reagent shouldgive rise to a readily detectable signal, e.g. a strongly-coloured areaeasily detectable by the eye. Thus, an important preferred embodiment ofthe invention is the use of “direct labels”, attached to one of thespecific binding members. Direct labels are well known in the art andhighly advantageous for their use in rapid diagnostic systems. Examplesof direct labels include, but are not limited to metal sols, non-metalsols, dye sols, latex particles, carbon sol, and liposome containedcolored bodies. Some of their advantages are that they can be used toproduce a visually detectable signal without the need to add furtherreagents, are readily visible to the naked eye without the aid ofinstrumentation, and can be readily used in a diagnostic device sincethey are stable when stored in the dry state. With respect to thelatter, their stability and immediate release on contact with a bufferreagent can be accomplished by the use of soluble glazes. In view of theabove comments, indirect labels, such an enzymes, e.g. alkalinephosphatase and horseradish peroxidase, are less preferred because theyusually require the addition of one or more substrates before a visiblesignal can be detected.

Non-metal sols, such as those of selenium, tellurium and sulfur may beproduced according to the methods described in U.S. Pat. No. 4,954,452(Yost, et al). Dye sol particles may be produced as described by Gribnauet al., in U.S. Pat. No. 4,373,932 and May et al., WO 88/08534, dyedlatex as described by May, supra, Snyder, EP-A 0 280 559 and 0 281 327,and dyes encapsulated in liposomes by Campbell et al., U.S. Pat. No.4,703,017. The use of polymerized dye materials in colloidal form forspecific binding assays is also described by in U.S. Pat. No. 4,166,105by Hirschreid which relates to labeled specific binding reagentsreactive with specific antigens prepared by linking fluorescent dyemolecules to analyte specific antibodies through polymers comprisingreactive functional groups. Also of interest is U.S. Pat. No. 4,313,734by Leuvering relating to metal sols; Leuvering, et al., “Sol ParticleImmunoassay (SPIA)”, Abstract, Journal of Immunoassay, 1(1), pp. 77-91(1980); Leuvering Dissertation (1984), Sol Particle Immunoassay (SPIA):The Use of Antibody Coated Particles as Labeled Antibodies in VariousTypes of Immunoassay; Uda et al., Anal. Biochem. 218 (1994), 259-264,DE-OS 41 32 133, page 3, lines 16-18, for applications as markers andTang et al., Nature 356 (1992), 152-154; Eisenbraun et al., DNA and CellBiology 12 (1993), 791-797. Furthermore it is also known thatnon-metallic colloidal particles such as carbon particles (vanAmerongen, Anabiotic '92 (1993), 193-199) can also be used. Moeremans,et al., EPO Application No. 158,746 discloses the use of colloidal metalparticles as labels in sandwich blot overlay assays. At presentcolloidal gold particles are used most frequently.

Among the direct labels, metallic sols are preferred, more preferablygold sol particles such as those described by Leuvering in U.S. Pat. No.4,313,734. Leuvering discloses the use of metal sol particles as labelsfor in vitro determination of immunological components in an aqueoustest medium. Specifically disclosed are immunoassay test kits for thedetection of antigens or antibodies employing one or more labeledcomponents obtained by coupling the component to particles of an aqueoussol dispersion of a metal, metal compound or polymer nuclei coated witha metal or metal compound having a particle size of at least 5 nm.

The metal sol particles to be used in accordance with the presentinvention may be prepared by methods which are well known in the priorart. For instance, the preparation of gold sol particles is disclosed inan article by G. Frens, Nature, 241, 20-22 (1973). Additionally, themetal sol particles may be metal or metal compounds or polymer nucleicoated with metals or metal compounds, all as described in the Leuveringpatent mentioned above. In this regard, the metal sol particles may beof platinum, gold, silver or copper or any number of metal compoundswhich exhibit characteristic colors.

5.2 Colloidal Gold Particles

Colloidal particles which are suitable as labels according to theinvention include those which may be conjugated to specific bindingmembers or general marker proteins without interfering with the activityof such reagents or with other reagents or analytes.

Colloidal metal particles are particularly suitable as labels accordingto the present invention and include those particles which are comprisedof metals or metal compounds selected from the group consisting of themetals platinum, gold, silver and copper and the metal compounds, silveriodide, silver bromide, copper hydroxide, iron oxide, iron hydroxide orhydrous oxide, aluminum hydroxide, or hydrous oxide, chromium hydroxideor hydrous hydroxide, lead sulfide, mercury sulphide, barium sulphateand titanium dioxide. Preferred colloidal metal particles include thosemade up of gold.

Colloidal gold particle markers are simple to use in comparison to otherconventional markers. For example, they do not require instrumentsnecessary for detection of other markers such as radioactive isotopesand unlike enzymes, they do not require the additional step of adding asubstrate.

Colloidal gold particles may be produced according to methods generallyknown in the art. Of interest to the present invention are thosereferences relating to the use of dispersions of colloidal particles inimmunological assay procedures. Specifically, Frens, Nature, 241, 20-23(1973) discloses methods for the production of gold sol particles ofvarying sizes through the reduction of gold chloride with aqueous sodiumcitrate. The colors of the visually detectable signal from the metalparticle label is dependent upon the identity and particle size of themetal particle which may be controlled by varying the concentration ofthe reactants. For example, colloidal gold particles produce colorsranging from orange to red to violet depending upon the particle size ofthe sol.

The colloidal gold reagent is selected for its unusual propertiesincluding the ability to intensify color to the naked eye whenconcentrated on solid surfaces, to minimally bind nonspecifically tosolid surfaces, to be prepared in relatively uniform particle sizes, andto be easily lyophilized and resolubilized. Colloidal gold particles canbe prepared in a number of ways through the reduction oftetrachloroauric acid which produces a variety of particle sizes rangingfrom 5 nm to 100 nm. The preferred particle sizes are from 15 to 20 nm.The colloidal gold particles can have an intermediary binder absorbed toits surface prior to the addition of the binding substance, but directattachment is satisfactory. Absorbing the selected binding substance isachieved by carefully controlling concentrations, ionic strength and pHof the reaction mixture. The choice of method of producing the colloidalgold raw material or the method of attaching the binding substance arewell known to those skilled in the art. After the labeling withcolloidal gold is complete, the reagent is differentially centrifuged orfiltered to control particle size. Particle sizing by gel filtrationmethods are also well known. The colloidal gold labeled reagent can beused as a colloidal suspension or as a lyophilized reagent with orwithout the presence of the aforesaid solid phase particles as anindicator reagent.

The resulting coated and stabilized colloidal metal particles may thenbe conjugated with various proteins. Any protein which may be subjectedto freeze-drying or other forms of drying such as by incubator,air-drying and spray drying may be applied in the present invention.Exemplary of protein for use in the present invention includes, but isnot limited to, polyclonal or monoclonal antibodies, antigen, lectin,protein A, protein G, bacterial, and the like. In those instance wherean immunodiagnostic assay is a sandwich format employing an antibody asthe capture reagent for the detection of an antigen as the targetanalyte, the binding member of the indicator reagent is usually a secondantibody having specificity for antigen bound to the first antibody, butwhich binds to the antigen at a site apart from where the first antibodyis bound. On the other hand, the binding member of the indicator reagentis usually an analogue, or other authentic example, of the antigen whichcan bind to the capture reagent at the same site where the targetanalyte binds in the case of a competitive format.

For details and engineering principles involved in the synthesis ofcolored particle conjugates see Horisberger, Evaluation of ColloidalGold as a Cytochromic Marker for Transmission and Scanning ElectronMicroscopy, Biol. Cellulaire, 36, 253-258 (1979); Leuvering et al, SolParticle Immunoassay, J. Immunoassay 1 (1), 77-91 (1980), and Frens,Controlled Nucleation for the Regulation of the Particle Size inMonodisperse Gold Suspensions, Nature, Physical Science, 241, pp. 20-22(1973). Surek, et al., Biochem. and Biophys. Res. Comm., 121, 284-289(1984) discloses the use of protein A labeled colloidal gold particlesfor the detection of specific antigens immobilized on nitrocellulosemembranes.

5.3 Drying Process—Sugar/Glazing Treatment

According to one important aspect of the invention, the label zone ofthe dried indicator reagent delivery unit essentially comprises theindicator reagent impregnated and dried within the thickness of a porousmaterial which can then be resolubilized by addition of themultifunctional buffer. Thus, by incorporating one of the assay reagentswithin the device of the present invention, makes possible the reductionin the number of steps required in the assay protocol by eliminating theaddition and/or prior mixing of an indicator reagent.

In order to assist the free mobility of the indicator reagent when thelabel zone of the dried indicator reagent delivery unit is moistenedwith the multifunctional buffer, the dried indicator reagent deliveryunit is pre-treated with a glazing material in the region to which theindicator reagent is applied. Glazing can be achieved, for example, bydepositing an aqueous sugar or cellulose solution, e.g. of sucrose orlactose, on the relevant region of the dried indicator reagent deliveryunit, while avoiding the remainder of the filter unit, and air drying.The indicator reagent can then be applied to the glazed portion.

The glazing process involving the use of one or more sugars (e.g.glucose, lactose, trehalose and sucrose) is highly advantageous whenemploying the dried indicator reagent of the present invention in thatthe sugar serves (1) as a protein stabilizer, (2) to improve the longterm stability of the dried indicator reagent, and (3) acts as a rapidreleasing agent. According to a preferred embodiment of the invention,sucrose was determined to be the best sugar compared to others in theperformance of the assay because of (1) its solubility, (2) short periodof drying, (3) the overall sensitivity of the assay result, (4) its useas a preservative, and (5) it is economical to use.

6.0 BUFFER REAGENT

Conventional diagnostic assays usually necessitate the use of two ormore fluid reagents in order to perform various steps of the assayprotocol including, for example, resolubilizing a dried indicatorreagent, diluting a fluid test sample, blocking the membrane surfacewhere the assay reaction takes place, facilitating transport of criticalreagents and/or washing unbound reactants from the reaction zone. Sinceeach of these steps involves the mixing or preparation of differentreactants, different formulations of liquid reagents are likely requireddue to differing pH, ionic strength, additives, type and strength ofbuffer, temperature, etc. For example, the resolubilization processusually requires the use of a physiological buffer such as bufferedsaline or double distilled water, the blocking process uses a liquidreagent formulated with any number of animal serum albumins, gelatin ornon-fat milk, and the washing and/or diluting process involves the useof a phosphate buffered saline containing different amounts ofsurfactant or detergent at neutral pH to remove any non-specific bindingreactants. Moreover, in order to ensure that the user performs each stepof the assay correctly using the appropriate liquid reagent, thereagents themselves must be clearly labeled and readily distinguishedfrom one another, so as to avoid any possible confusion and user error.

An important aspect of the present invention overcomes the variousproblems described above associated with the use of several assayreagents by providing a multifunctional buffer for single utilization inthe 2-step assay procedure. The multifunctional buffer is formulated toserve as a combination resolubilization reagent of the dried indicatorreagent, transport facilitating reagent of resolubilized indicatorreagent from the label zone of the dried indicator reagent delivery unitto reaction zone of the test unit, and washing reagent to remove unboundreactants from the reaction zone. In order to simplify the number ofreagents and steps required to perform the assay, the multifunctionalbuffer has been specially formulated to be used in conjunction with thedried indicator reagent. It is therefore, particularly advantageous toutilize the multifunctional buffer and dried indicator reagent as acombined system since the multifunctional buffer allows optimalsensitivity and higher specificity to be achieved during performance ofthe assay, while additionally avoiding aggregation and inactivation ofthe dried indicator reagent in solution.

As will be apparent to one skilled in the art, the composition of themultifunctional buffer may vary in accordance with the requirements ofthe specific assay such as the particular capture reagent and indicatorreagent employed to determine the presence of a target analyte in a testsample, as well as the nature of the analyte itself. In general, themultifunctional buffer will contain compounds that have primaryfunctions in the assay with respect to their properties in serving as adiluting, washing and resolubilizing agent. However, since the reactionzone of the present invention is already pretreated with conventionalblocking agents following immobilization of the capture reagent, thebuffer formulation eliminates the need to include a non-specificblocking agent. A method of using the multifunctional buffer as providedby the present invention essentially involves dropwise addition of thebuffer to the dried indicator reagent delivery unit in the final step ofthe 2-step assay to resolubilize the dried indicator reagent. A kitcontaining the multifunctional buffer as a component is also provided.

Accordingly, the present invention provides an improved buffer whichserves as a multifunctional reagent without sacrificing either thesensitivity or specificity of the assay comprising: (1) a biologicalbuffer to maintain the pH between about 7.0 to 10.0; (2) at least onesurfactant to reduce non-specific binding of assay reagents whilesimultaneously avoiding inhibition of a specific binding interaction;(3) a high molecular weight polymer as a dispersing and suspendingreagent having a molecular weight in a range of from about 2×10² toabout 2×10⁶ D; (4) a pH stabilizer to maintain the pH of themultifunctional buffer between about pH 7.0 to 10.0; (5) an ionic saltto reduce the non-specific binding of antibodies; (6) at least onepreservative to reduce bacterial and microbial growth; and (7) a calciumchelator to prevent a whole blood test sample from clotting; wherein thebiological buffer, surfactant, high molecular weight polymer, pHstabilizer, ionic salt, preservative and calcium chelator are all ateffective concentrations.

The improved multifunctional buffer composition of the invention caninclude a conventional buffer such as a phosphate buffer, MES(morpholino-ethanesulfonic acid) buffers, BIS-TRIS buffers, citratebuffers, TRIS-HCl buffers and borate buffers, at an effectiveconcentration which can range from about 5 to 100 mM, preferably in therange of from about 5 to 30 mM, and most preferably about 5 mM. Thepreferred buffer is a phosphate buffer, preferably comprising sodiumphosphate, monobasic and sodium phosphate, dibasic, at concentrationssuch that the effective pH of the buffer is achieved. The pH of thebuffer of the present invention can range from a pH of about 7.0 to a pHof about 10.0.

The biological detergents (surfactants) used in the present inventioncan include non-ionic surfactants, anionic surfactants, zwitterionicsurfactants and cationic surfactants. The Non-ionic detergents useful inthe invention include polyoxyethylene sorbitan monolaurate (Tween® 20),polyoxyethylene sorbitan monooleate (Tween® 80), polyoxyethylene ethers(Triton®, Brij®) and octylphenel ethylene oxide (Nonidet®). Preferably,non-ionic detergents are used. The most preferred non-ionic detergent isTriton® X-100. Non-ionic detergent acts as a dispersing agent to reducethe non-specific binding of antibodies/antigens to the reaction membranewhich may occur as a result of target analyte adhering to the solidphase due to a non-specific reaction, thereby increasing the backgroundof the assay. Although biological detergents reduce the event of suchbinding caused by nonpolar or hydrophobic interactions, non-ionicdetergents are preferred for their ability to reduce non-specificbinding while avoiding the inhibition of specific binding. Effectiveconcentrations of the biological detergent range from about 0.01 toabout 0.50% (w/v), preferably range from about 0.05 to about 0.10%(w/v), and most preferably the concentration is about 0.07% (w/v).

Ionic salts provide a source of cations and anions which helps to reducethe frequency of non-specific binding of antibodies, other than analyteantibodies, caused by ionic interactions. Salts that are useful in theformulation of the multifunctional buffer reagent are NaCl and KCl, mostpreferably NaCl. Effective concentrations of sodium chloride range fromabout 0 to about 300 mM, preferably range from about 50 to about 200 mM.

The high molecular weight polymer functions as a dispersing andsuspending reagent while additionally preserving the binding capacity ofantibodies. Examples of high molecular weight polymers which may be usedin the buffer are polyvinylpyrrolidone (PVP), dextrans, polyethyleneglycol (PEG), and polyvinyl alcohol, to name a few. The preferred highmolecular weight polymer for use in generating the buffer is PVP; mostpreferably PVP-40, at an effective concentration. Effectiveconcentrations of PVP in the buffer of the invention range from about0.1 to about 3.0% (w/v), preferably range from about 0.5 to about 2.5%,and most preferably the concentration is about 1.4%. The high molecularweight polymer selected for use in the invention can include PVP havingmolecular weights of from about 10 kD to about 1500 kD, dextrans withmolecular weights ranging from about 10 kD to about 2000 kD,polyethylene glycols (PEG) having molecular weights in the range of fromabout 200 D to about 10,000 D, and polyvinyl alcohol having a molecularweight of about 10,000 D to about 100,000 D. Other examples of highmolecular weight polymer also include polybrene(hexadimethrine bromide),methylcellulose, gum acacia, protamine sulfate, merquat, celquat andmagnafloc, provided at an effective concentration.

It is preferable to include a calcium chelating agent, such asethylenediaminetetraacetic acid (EDTA), or salts thereof, in themultifunctional buffer composition, to reduce or prevent the possibleclotting of a finger-pricked whole blood test sample through the bloodcoagulation process. Calcium chelating agents, other than EDTA, such ascitrate, citrate salts, and ethylenebis(oxyethylenenitrilo)tetraaceticacid may similarly be used. Biopolymers (i.e. non-chelating agents) suchas heparin and sulfated chitosan, which will inactivate specificclotting factors within the blood coagulation process can also be used.EDTA is included in the buffer composition at an effective concentrationranging from about 5 mM to 100 mM, more preferably at about 10 mM toabout 50 mM, and most preferably about 20 mM.

The pH stabilizer functions to maintain the pH of the buffer within arange of about pH 7.0 to 10.0. An exemplary pH stabilizer includestrizma hydrochloride, although other known stabilizers may also beuseful in this composition. The effective concentration of trizmahydrochloride is preferably from about 20 to 30 mM.

7.0 HOUSING

In general, the assay composite comprising the test unit and the driedindicator reagent delivery unit can be housed in a suitable container toform an analytical apparatus. Preferably, the container should safeguardthe solid phase materials and dried indicator reagent from contaminationand to provide ease and convenience in handling of the assay device.Moreover, the container should be leak-proof thereby ensuringcontainment of fluids and their safe disposal after use.

The apparatus 13 illustrated in FIGS. 4 and 5 provides a representativeexample of the type of container that can be included in a test kitwhich incorporates the flow-through device of the present invention. Theapparatus 13 comprises two detachable components, namely the testcartridge 14 and the dried indicator reagent delivery cap 15, which arevertically and spatially distinct to one another when placed intransient fluid communication during the assay protocol. The housing iscapable of maintaining the layers of the test unit under compression soas to provide continuous and uniform contact therebetween and so thatliquid will flow uniformly through the apparatus 13. The housing will bemade of an inert material conveniently being any of a variety ofdisposable commercial plastics which may be molded, for example,polyethylene, polypropylene, styrene, ABS, polyacrylate, polystyrene, orthe like.

Although the two components of the apparatus 13 have the particularconfiguration and dimensions depicted therein, any other appropriatedesign or modifications may be employed so long as the components arestill capable of being transiently connected to one another in a singlemovement during the assay protocol. The means of connecting the twocomponents is not critical so long as so that they are properly alignedto effect optimal fluid communication with one another uponinterconnection of the dried indicator reagent delivery cap 15 with thetest cartridge 14. For example, according to the design shown in FIGS. 4and 5, the dried indicator reagent delivery cap 15 may be frictionallyfitted to the reservoir 22 of the test cartridge 14. Although notillustrated therein, the dried indicator reagent delivery cap 15 may beoptionally hinged to the test cartridge 14 to avoid possible lost ormisplacement of the two components. On the other hand, the twocomponents could be slidably and reversibly disposed to one another in asingle horizontal movement providing the dried indicator reagentdelivery unit is engaged in proper alignment above the test unit. Inthis particular instance, proper alignment of the two components may beachieved through the use of guide rails, or projections designed toalign with recesses formed in the device or housing, additionally actingas an interconnection means for the two components.

The precise dimensions of the housing are not essential to the functionof the assay apparatus, but in general, the apparatus will be of a sizeconvenient for transport, manipulation, and assembly. The housing willgenerally have a length in the range of about 2 to 5 cm, preferably 3.5cm. The width will be in the range of about 1 to 3 cm, preferably 2.5cm. The height of the housing will be in the range of about 0.5 to 5 cm,preferably 1.3 cm.

FIG. 4 provides an exploded view of the apparatus 13 comprising the testunit 2 and the dried indicator reagent delivery unit 3, while FIG. 5provides an enlarged vertical cross-sectional view of the fullyassembled apparatus 13. The apparatus 13 of the present inventioncomprises two separate components in its fully assembled form, namelythe test cartridge 14, which contains the test unit 2, and the driedindicator reagent delivery cap 15, which contains the dried indicatorreagent delivery unit 3. The test cartridge 14 and the dried indicatorreagent delivery cap 15 are designed to be connected to one anotherbriefly during the assay protocol. The apparatus 13 is intended to besimple in design and construction, and can be manufactured using readilyavailable materials.

As shown in FIG. 4, the test cartridge 14 of the apparatus 13 houses thetest unit 2 which comprises both a top member 16 and a bottom member 17.The outer perimeter of the bottom member 17 has a slightly indentedridge 18 which allows it to be fitted and interconnected with the rim 19bordering the top member 16 to form the assembled test cartridge 14. Itwill be appreciated by those skilled in the art that while the testcartridge 14 shown in FIGS. 4 and 5 has a rectangular shape, it is notlimited to this particular configuration so long as it can be adapted tohold the absorbent material, or pad 20, in direct contact with thereaction membrane 21.

Contained within the top member 16 of the test cartridge 14 is areservoir 22 which is in direct alignment with the exposed reactionmembrane 21 of the test unit 2. The reservoir 22 (a) provides access tothe reaction zone for introducing the fluid test sample, (b) providesoperable attachment of the dried indicator reagent delivery cap 15 forintroduction of the multifunctional buffer reagent, and (c) permitsviewing of the test result on the reaction membrane 21 following removalof the dried indicator reagent delivery cap 15, i.e. detect the color,or fluorescence, or other signal, in the indicator zone(s). As depictedin the drawing, the upper surface surrounding the reservoir 22 isslightly curved and extended downwards so as to form a cup-likereceptacle terminating at a portion of the reaction membrane 21. In thisway, the amount of test sample introduced into the reservoir 22 cannotbypass any components of the apparatus 13. The configuration of theinner wall 23 and the dimensions of the reservoir 22 are selected sothat the reservoir 22 can connect to and be in operable association withthe dried indicator reagent delivery cap 15 during the assay protocol.Preferably, both the reservoir 22 and the dried indicator reagentdelivery cap 15 have a funnel shape configuration. Thus, when thereservoir 22 and the dried indicator reagent delivery cap 15 are in theoperating position and the multifunctional buffer is applied to thefilter cap 15, this configuration will permit a suitable amount of thebuffer to contact and pass through a small amount of surface area of thereaction membrane 21. Thus, by selectively matching the size ofreservoir 22 with the dried indicator reagent delivery cap 15, theoperation of the apparatus 13 can be simplified so that, for example,the multifunctional buffer 12 can be delivered to the reservoir 22 in asingle step of the assay procedure.

According to the embodiment shown in FIGS. 4 and 5, the dried indicatorreagent delivery cap 15 is detachable affixed to the reservoir 22 of thetest cartridge 14 by means of a friction fit between the inner wall 23of the reservoir 22 and the external wall 33′ of the filter cap 15. Suchother means for detachably affixing the dried indicator reagent deliverycap 15 to the test cartridge 14 can be used. In addition, the height ofthe external wall 33′ of the dried indicator reagent delivery cap 15 isslightly less than the height of the inner wall 23 of the reservoir 22so that when the filter cap 15 is affixed to the reservoir 22, the base24 of the filter cap 15 terminates immediately above, but not touching,the reaction membrane 21. The dimensions of both the reservoir 22 andthe dried indicator reagent delivery cap 15 can be varied withoutaffecting the performance of the apparatus 13, although the followingapproximate dimensions have been determined as satisfactory: reservoir22—1.5 cm top and bottom diameters and 0.6 cm deep; dried indicatorreagent delivery cap 15—0.9 cm bottom diameter, 1.1 cm top diameter, and0.5 cm deep.

As described above, the test unit of the present invention comprises areaction membrane 21 and an absorbent pad 20, whereby the lower surfaceof the reaction membrane 21 is supported by the upper surface of theabsorbent pad 20. The reaction membrane 21, which contains capturereagent capable of binding target analyte, essentially defines thereaction zone in which various specific binding reactions take placeduring the assay. As previously described, the reaction membrane 21 canbe fabricated from a number of biologically inert, porous materials.

Positioned directly underneath the lower surface of the reactionmembrane 21, and in fluid communication therewith, is an absorbent pad20 defining the absorbent zone. In embodiments of the invention whereease of manufacture and reduced costs are desired, the entire uppersurface of the absorbent pad 20 is typically immediately adjacent thelower surface of the reaction membrane 21. The test unit may optionallyinclude a separating means between the reaction membrane 21 and theabsorbent pad 20 which will generally be incapable of binding the targetanalyte of interest. According to the embodiment shown in FIG. 4, theseparating means in the form of a spacer layer 25 isolates a portion ofthe reaction membrane 21 from the absorbent pad 20. Although notcritical to the performance of the apparatus 13, the spacer layer 25serves to secure the reaction membrane 21 in place and permit assayreagents to flow uniformly from the upper surface down to the lowersurface of the assay apparatus 13.

The spacer layer 25 has an opening 26 defined by a rim 27 which hasperimeter dimensions and a shape similar to the reaction membrane 21thereby enabling the upper and lower surfaces of the reaction membrane21 to be accessible when the membrane 21 and the spacer layer 25 aresealed together to form a press-fit piece. Referring to FIG. 4, whichdepicts one embodiment of the apparatus 13, a portion of the reactionmembrane's 21 upper surface is fully exposed so that when the diagnosticassay is performed, the fluid test sample and the assay reagents can beadded directly to the reaction membrane 21. The reaction membrane 21 issized to completely cover the opening 26. Preferably the reactionmembrane 21 will be the same shape as the opening 26, but sized slightlylarger than the opening 26 so that it can be sealed to the lower surfaceof the spacer layer 25 at the periphery of the opening 26. However, theshape of the reaction membrane 21 and the shape of the opening 26 candiffer and are not limited to the configuration shown in FIG. 4. Thus,in combination, the rim 27 surrounding the opening 26 and the exposedupper surface of the reaction membrane 21 essentially define a testregion. Moreover, after the test cartridge 14 of the apparatus 13 isassembled, the absorbent pad 20 is still capable of contacting the lowersurface of reaction membrane 21 located directly beneath the reactionmembrane 21. The dimensions of the spacer layer 25 and the absorbent pad20 are chosen to fit cooperatively within the base of the test cartridge14, thereby ensuring that the absorbent pad 20 is in proper alignmentand fluid communication with the lower surface of reaction membrane 21.Generally, the surface area of the upper surface of the absorbent pad 20will usually be greater than that of the reaction membrane 21, butsimilar to that of the spacer layer 25.

The absorbent pad 20 is selected to have a capillary pore size so as toinduce flow of the fluid test sample through the reaction membrane 21without the use of external means. Thus, conveniently, the absorbent pad20 serves to both promote and direct the flow of reagents through thereaction membrane 21. The absorbent pad 20 is of sufficient size andcomposition so that it is capable of absorbing excess sample, indicatorreagent and buffer. The material from which the absorbent pad 20 isfabricated may be any permeable wettable material that is substantiallyinert to the assay reagents employed in the performance of an assay. Theabsorbent pad 20 will have essentially the same perimeter dimensions andshape as the spacer layer 25 which holds the reaction membrane 21. Theprecise thickness of the absorbent pad 20 is not essential to thefunction of the present invention, generally ranging from about 2 to 10mm.

The second component of the apparatus is the funnel-shaped driedindicator reagent delivery cap 15 which readily accommodates a suitableamount of the multifunctional buffer needed to perform the assay in asingle application. The dried indicator reagent delivery cap 15comprises the dried indicator reagent delivery unit 3 and inner 28 andouter 29 sleeves being open-ended at both the top and bottom. The bottomopening 30, 30′ of sleeves 28 and 29 is sized to achieve the flow ratedesired for the assay in question. The opening of the sleeves canconveniently have a diameter in the range of 12.6 to 15.2 mm. Preferablythe opening 30, 30′ diameter is 9.5 mm.

In the assembled form, the dried indicator reagent delivery cap 15comprises a funnel 31 having at its top outwardly extending flanges 32,32′ and depending sidewalls 33, 33′. The depending sidewalls 33 of theouter sleeve 29 terminate at base 24. The opening 30′ at the base 24allows a stream of fluid traveling through the funnel 31 to flow intothe test cartridge 14. The dried indicator reagent delivery unit 3 ofthe present invention is securely held in the base 24 of dried indicatorreagent delivery cap 15 by the inner 28 and outer sleeves 29 of thedried indicator reagent delivery cap 15. An inner collar 34, integrallyformed at the base 24 of the outer sleeve 29, is capable of supportingthe dried indicator reagent delivery unit 3 so that when the innersleeve 28 is frictionally fitted inside the outer sleeve 29, the driedindicator reagent delivery unit 3 will be held permanently in place.

The dried indicator reagent delivery unit 3 comprises a filter mediumimpregnated with dried indicator reagent which defines the label zone.The dried indicator reagent is resolubilized and transported by themultifunctional buffer to the reaction membrane 21 following addition ofthe buffer to the dried indicator reagent delivery cap 15. The selectionof the filter medium for the dried indicator reagent delivery unit 3 isnot critical to the invention and can be any suitably absorbent, porousor capillary possessing material through which the multifunctionalbuffer and resolubilized indicator reagent may be transported by wickingaction. The criteria of selection is that the material allow for theresolubilization and mixing of the dried indicator reagent upon additionof the multifunctional buffer, as well as initiate the transfer of thebuffer and freshly dissolved indicator reagent to the reaction membrane21 of the test unit 2.

For convenience of manipulation in using the apparatus 13, a handle 35is secured to the extending flange 32 of the dried indicator reagentdelivery cap 15 so that when the filter cap 15 is affixed to thereservoir 22, it extends slightly beyond the boundary of the reservoir22 for ease of removal of the dried indicator reagent delivery cap 15from the test cartridge 14.

A representative example of a modified version of the test cartridge ofthe invention incorporating a blood separation zone in lateral fluidcommunication with the reaction zone for the detection of analyte in awhole blood sample is illustrated in FIGS. 7A and 7B.

As shown in FIG. 7A, the test cartridge is provided with a top member 16constructed and adapted to fit snugly with a bottom member 17. In thisparticular embodiment, the top member 16 of the test cartridge defines afirst opening or internal recess therethrough in the form of a reservoir22. The reservoir 22 serves to (a) provide operable attachment of thedried indicator reagent delivery cap for introduction of themultifunctional buffer reagent, and (b) permit viewing of the testresult on the membrane following removal of the dried indicator reagentdelivery cap, i.e. detect the color or fluorescence, or other signal, inthe indicator zone(s). Thus, the configuration and dimensions of thereservoir 22 are selected on the basis that it can be operably connectedto the dried indicator reagent delivery cap to enable transient fluidcommunication between the label zone of the dried indicator reagentdelivery unit and the reaction zone of the test unit.

Spaced a short lateral distance from the reservoir 22, the top memberdefines a second opening therethrough in the form of a reservoir 104which may, as shown, have beveled sides, or may be in any shape or sizeor configuration of convenience which will sufficiently direct andprovide access to the first end of the blood separation zone uponapplication of a whole blood sample. After introducing a whole bloodsample to the reservoir 104 and allowing for a short incubation time toenable sufficient separation and migration of the RBC-free fluid alongthe blood separation zone to the reaction zone, the dried indicatorreagent delivery cap is operably attached to the reservoir 22 to enablecompletion of the 2-step assay protocol so that a final determinationfor the presence of target analyte can be made.

As shown in FIG. 7B, the bottom member 17 of the test cartridge providesa first base structure 105 having a plurality of supporting walls whichserve as a solid enclosure for the absorbent pad and thus, is configuredto receive and hold the absorbent pad securely in place. Additionallyprovided is a second base structure 106 having a plurality of protrudingcolumns of the same height which serves as an elevated support for theblood separation zone. The position of the second base structure 106 inrelation to the first base structure 105, as well as its configuration,are such that when the blood separation zone is positioned within thebottom member 17, the blood separation zone is contiguous with and indirect planar horizontal alignment with the reaction zone. Although thebase structure 106 depicted therein has a plurality of supportingcolumns and/or walls which serve to support the perimeter and centre ofthe blood separation zone, any number of configurations or strategiesare possible as long as the blood separation zone is securely andcorrectly positioned in relation to the reaction zone when the testcartridge is fully assembled.

8.0 METHODOLOGY

In operation, the apparatus of the present invention broadly is used todetermine the presence of target analyte in a fluid test sample,employing at least one capture reagent to form a detectible product onthe reaction membrane as an indication that the analyte is present inthe sample. The assay device and apparatus is particularly applicable toan immunoassay wherein the sample component is one component of animmunological pair including antigens, antibodies, or haptens. Theimmunological pair includes two components which immunologically bind toeach other. Specific immunological pairs include antigens and theirantibodies (monoclonal antibodies or affinity purified polyclonalantibodies, including fragments thereof), or biologically functionalhaptens and their antibodies. While monoclonal antibodies have knownadvantages over polyclonal antibodies, either type of immunologicalreagents can be used in accordance with the present invention. Thus, forsimplicity of representation, the assay and device of the presentinvention will be described with respect to immunoassays using theantigen-antibody immunological pair. The fluid test sample isbiologically derived, e.g. urine or serum, and the capture reagent andindicator reagent can comprise an antibody or antigen, depending on theanalyte of interest and whether the sandwich or competitive technique isemployed.

The immunoassays that use the analytical apparatus of the presentinvention can be very simple and fast, and can be qualitative orsemi-quantitative. The analytical apparatus can be adapted for use inmany different types of assays. For example, the target analyte can be ahormone, antibody, antigen, protein, etc. A non-inclusive list ofpossible target analytes is provided in U.S. Pat. No. 5,006,464 (Chu, etal.). The immunoassay format, will depend on the target analyte soughtto be detected. Again, these are already known in the art. It will beappreciated by those skilled in the art that in order to maximizesensitivity for the detection of a particular target analyte, variouscomponents of the analytical device and/or assay procedure can bemodified, such as the porosity, thickness and type of material used forthe reaction membrane. The analytical device used in the assaysessentially requires no sample manipulation and the entire assayprotocol can be performed in less than 1 minute.

The assay device of the invention is contemplated to be used in anyflow-through immunoassay procedure including competitive and preferablysandwich assays. As mentioned above, the reaction membrane is coatedwith a capture reagent, generally a specific antibody, or fragmentthereof. Alternatively, if the target analyte is an antibody, thecapture reagent may be a specific antigen. In either case, after capturereagent is applied to the membrane, it is preferred to fill anyunoccupied binding sites with an inert protein to prevent nonspecificbinding of any other assay reagent, such as the indicator reagent, tothe membrane. In the present disclosure, the term “inert protein” meansa protein which is immunologically unreactive toward any other componentof the assay and which does not substantially bind nonspecifically toother proteins in the assay medium, with the understanding that theinert protein may well be immunologically reactive toward othermaterials which are not part of the assay of the invention.Representative non-limiting examples of suitable inert proteins arealbumin and casein.

Referring first to a sandwich assay for the detection of antigen, thecapture reagent will be a first antibody specific for the predeterminedantigen and the indicator reagent will comprise a second antibody alsospecific for the predetermined antigen. The first antibody, preferably amonoclonal antibody or an affinity purified polyclonal antibody, isbound to the reaction membrane as the capture reagent. The firstantibody is selected for its ability to recognize and bind to a specificepitopic site on the antigen of interest. The second antibody formingpart of the indicator reagent will be selected on the basis that it willrecognize and bind to an alternative epitopic site on the antigen ofinterest and thus, not interfere with the binding interaction of thefirst antibody with the antigen. One of skill in the art will alsoappreciate that the sandwich assay may also be performed by reversingthe roles of the antigen and antibody. For example, the immobilizedmember of the immunological pair may be the antigen for the detection ofan antibody of interest in the test sample utilizing a labeledanti-human antibody or Protein A as the indicator reagent.

Referring to FIGS. 4, 5 and 7, and using a fluid test sample other thanwhole blood, the method of the invention is carried out by directlydepositing a volume of the fluid test sample onto the upper surface ofthe reaction membrane 21 by introduction through the opening in thereservoir 22. The amount of fluid test sample and multifunctional bufferreagent added to the assay apparatus 13 via the reservoir 22 may varywith different embodiments of the subject invention. In general, for agiven specific embodiment, a predetermined and undiluted quantity oftest sample will be added, while the multifunctional buffer reagent willnormally be added in excess. A predetermined volume of sample ispreferably added dropwise, using a standard sterilized pipette, to thecentre of the reservoir 22 so that uniform contact between the sampleand the immobilized capture reagent is maintained. In a preferredembodiment of the invention, only a single drop of the fluid test sampleis required to be added to the reservoir. As the fluid test sample isinduced to flow through the absorbent pad 20 by capillary action, anyfree antigen that may be present in the test sample comes into contactwith the antibody immobilized to the surface of the reaction membrane21. The free antigen thus becomes immobilized by the antibody while thefluid test sample diffuses into the absorbent pad 20 underneath. Thedried indicator reagent delivery cap 15 is subsequently connected andbrought into operable association with the reservoir 22 of the testcartridge 14. A predetermined volume of multifunctional buffer may bemeasured by a marker in the reservoir 22, or preferably added dropwise,using a second standard sterilized pipette. The dropwise addition of thebuffer reagent to the centre of the post filter-cap 15 should encourageuniform contact and saturation of the dried indicator reagent deliveryunit 3 so that the dried indicator reagent will be fully resolubilized.Furthermore, the volume of buffer reagent should be sufficient toseparate any unbound indicator reagent from the reaction membrane 21after the specific binding interactions have occurred. As the stream ofbuffer reagent contacts and subsequently diffuses through the reactionmembrane 21, unbound reactants are separated from the bound reactants.According to a preferred embodiment of the present invention a range of10 to 15 drops of buffer can be added to the dried indicator reagentdelivery cap 15. Upon addition of the multifunctional buffer andfollowing resolubilization of the dried indicator reagent comprisinglabeled antibody, the labeled antibody is transported to the reactionmembrane 21 by the buffer, where it will bind with any antigen that isbound by the immobilized antibody. Due to the volume and chemicalproperties of the multifunctional buffer, a separate washing step is notrequired in order to remove unbound labeled antibody. The presence oflabeled antibody on the reaction membrane 21 is then determined as anindication of the presence of the target antigen in the sample. Thebinding reaction of the labeled antibody with the antigen produces avisually detectable signal indicative of a positive result that iseasily observed directly following removal of the dried indicatorreagent delivery cap 15.

The present invention is also applicable to the competitive bindingtechnique for example, described in U.S. Pat. No. 4,366,241 (Tom, etal.). In such system for the detection of antigen in a fluid testsample, the corresponding member of the immunological pair, namely theantibody, is immobilized on the reaction membrane 21 surface. However,the binding member of the indicator reagent will be an authentic sampleof the target antigen which has a comparable binding affinity for theantibody immobilized to the reaction membrane 21. After the fluid testsample is deposited on the reaction membrane 21, the presence ofantigen, if any, and the ancillary antigen of the indicator reagentcompete for sites of attachment to the antibody. Since the immobilizedantibody is in limited supply, a competition is set up between theantigen in the sample and the labeled antigen. If there is no antigenpresent in the test sample, labeled antigen aggregates on the reactionmembrane 21. Thus, the presence of color signifies a negative result dueto the absence of detectable levels of antigen in the sample. If antigenis present, no color develops due to a reduction in the amount oflabeled antigen bound by the immobilized antibody having binding sitesalready occupied by the target antigen, thus indicating a positiveresult. Accordingly, the signal emitted from the label is inverselyproportional to the amount of antigen in the sample. Moreover, as withthe sandwich assay, the competitive binding assay may be performed byreversing the roles of the antigen and antibody. For example, theimmobilized member of the immunological pair may be the antigen for thedetection of an antibody of interest in the test sample which competeswith labeled antibody.

As a further consideration, this system could be expanded to include thesimultaneous detection of two or more analytes of interest in a fluidtest sample by using a corresponding number of immobilized immunologicalreagents on the reaction zone. As an example, a first antibody may beselected that is reactive with a particular subunit of a number ofdifferent antibodies. If a second antibody is specific for a subunit ofone antigen only, such second antibody can be used as the immobilizedantibody and a single labeled first antibody can be used as theuniversal labeled antibody for antigen of interest. On the other hand,two different types of immobilized antibody may be employed if it isexpected that antigen capable of being recognized by its appropriatebinding partner is likely to present in a fluid test sample, e.g.anti-HCV antibody and anti-HIV antibody found in a co-infected patientsample.

In the case of analyzing a whole blood sample, the method of theinvention is essentially identical to that described above with theexception that a volume of the sample is directly deposited into thereservoir 104 which allows access to and direct contact with the firstend of the blood separation zone through the opening defined therein(refer also to FIG. 7A). The amount of the whole blood sample applied tothe reservoir 104 and the amount of multifunctional buffer reagent addedto the reservoir 22 may vary with different embodiments of the subjectinvention. In general, for a given specific embodiment, a predeterminedand undiluted quantity of test sample will be added, while themultifunctional buffer reagent will normally be added in excess. Apredetermined volume of the sample is preferably added dropwise, using astandard sterilized pipette, to the centre of the reservoir 104 so thatuniform contact between the sample and the blood separation zone isensured.

In a preferred embodiment of the invention, only two drops of the wholeblood sample are required to be added to the reservoir 104. Following ashort incubation period, the RBC-free portion of the whole blood sample,including any analyte, migrates in a lateral direction along the bloodseparation zone until it arrives at the reaction membrane 21. As theRBC-free fluid test sample is induced to flow through the absorbent pad20 by capillary action, any free antigen that may be present in the testsample comes into contact with the antibody immobilized to the surfaceof the reaction membrane 21. The free antigen thus becomes bound by theantibody while the fluid test sample, along with non-essentialcomponents, diffuses into the absorbent pad 20 underneath. The driedindicator reagent delivery cap 15 is subsequently connected and broughtinto operable association with the reservoir 22 of the test cartridge14. A predetermined volume of multifunctional buffer may be measured bya marker in the reservoir 22, or preferably added dropwise, using asecond standard sterilized pipette. The dropwise addition of the bufferreagent to the centre of the post filter-cap 15 should encourage uniformcontact and saturation of the dried indicator reagent delivery unit 3 sothat the dried indicator reagent will be fully resolubilized.Furthermore, the volume of buffer reagent should be sufficient toseparate any unbound indicator reagent from the reaction membrane 21after the specific binding interactions have occurred. As the stream ofbuffer reagent contacts and subsequently diffuses through the reactionmembrane 21, unbound reactants are separated from the bound reactants.According to a preferred embodiment of the present invention a range of10 to 15 drops of buffer can be added to the dried indicator reagentdelivery cap 15. Upon addition of the multifunctional buffer andfollowing resolubilization of the dried indicator reagent comprisinglabeled antibody, the labeled antibody is transported to the reactionmembrane 21 by the buffer, where it will complex with any antigen thatis bound by the immobilized antibody. Due to the volume and chemicalproperties of the multifunctional buffer, a separate washing step is notrequired in order to remove unbound labeled antibody. The presence oflabeled antibody on the reaction membrane 21 is then determined as anindication of the presence of the target antigen in the sample. Thebinding reaction of the labeled antibody with the antigen produces avisually detectable signal indicative of a positive result that iseasily observed following removal of the dried indicator reagentdelivery cap 15.

9.0 TEST KIT

According to the invention, kits may be produced which include the rapidassay device, the multifunctional buffer, as well as instructionsdescribing the assay protocol for determining the presence of a targetanalyte in a fluid test sample. The diagnostic device of the presentinvention, which incorporates the test unit and dried indicator reagentdelivery unit, will typically be packaged in the form of a diagnostickit for use in the detection of the target analyte of interest. The kitwill normally include the flow-through assay device, preferably housedin a suitable container, the multifunctional buffer, disposable plasticpipettes and instructions describing the method for carrying out theassay protocol. Depending on the type of assay performed, i.e. sandwichor competitive, and the target analyte to be determined in the fluidtest sample, the instructions will also include the relative amounts oftest sample and multifunctional buffer to be added to the test unit anddried indicator reagent delivery unit, respectively. In addition, thetime periods required involving the sequential addition of the sampleand buffer, as well as that the time required for the generation of aresult will be included.

The preferred kit of the present invention uses the flow-throughdiagnostic device as described and shown in FIGS. 1 to 3. Preferably,the flow-through diagnostic device is housed within a suitable containerthat can be included in the test kit comprising two detachablecomponents, each component separately containing the test unit and thedried indicator reagent delivery unit, which are arranged in avertically and spatially distinct format. In particular, the design ofthe container should allow the test unit and dried indicator reagentdelivery unit to be operably connected to one another so that thereaction zone and the label zone can be placed in transient fluidcommunication with one another during the assay protocol in a singlemovement. The container housing the test unit should be capable ofmaintaining the layers of the test unit under compression so as toprovide continuous and uniform contact therebetween so that liquid willflow uniformly through the apparatus. FIGS. 4, 5 and 7 provides arepresentative example of the type of container that can be included ina test kit which incorporates the flow-through device of the presentinvention.

The invention will be further understood from the following non-limitingexamples. The following examples are provided to describe in detail someof the representative, presently preferred methods and materials of theinvention. These examples are provided for purposes of illustration ofthe inventive concepts, and are not intended to limit the scope of theinvention as defined by the appended claims.

10.0 EXAMPLES

The foregoing is a general description of the apparatus, method andreagents of the invention. A sandwich-type reaction may be performed forthe detection of Helicobacter pylori. Thus, by way of an exampleprovided below, the capture reagent is a solution of Helicobacter pyloriwhich is applied to the reaction membrane of the test unit. Although dyesols, gold sols or coloured latex particles may be linked to Protein Ato form the indicator reagent, the preferred visual label utilized inthe example assay will be colloidal gold particles. Using an apparatuscomprising the test unit and dried indicator reagent delivery unit, suchas the one illustrated in FIGS. 4 and 5, and by performing the 2-steprapid assay of the present invention, a determination of antibodyagainst Helicobacter pylori in a serum test sample can be made in lessthan three minutes.

10.1 Preparation of the Diagnostic Assay Apparatus

The apparatus 13, illustrated in FIGS. 4 and 5 comprising a testcartridge 14 and dried indicator reagent delivery cap 15, represents asuitable container to house the test unit 2 and dried indicator reagentdelivery unit 3 of the present invention.

A. Test Cartridge

The test cartridge, which houses the test unit of the rapid test device,is made of clean technical grade white polypropylene plastic and has atop 16 and bottom 17 component. Both are made in synchronized 16 cavitymold, precisely engineered to allow a snugly fitting tight seal when thetwo components are pressed together. The components are supplied asindividual casings by Top View International Limited, Hong Kong, and areassembled at the manufacturing plant of MedMira Laboratories, Halifax,Canada. These components meet the following criteria:

Appearance—a clear, white smooth texture of the plastic.

A snug fit to produce a leak-proof housing to ensure safe containment ofall applied liquids.

Consistency of dimensions to specifications of 2.5 cm width, 3.5 cmlength, and 1.3 cm height.

Consistency of dimensions of the reservoir 22 opening of 1.6 cm indiameter and a formed cylinder depth of 0.5 cm.

Consistency in the location of the reservoir 22 in the top component ofthe test cartridge 14.

B. Reaction Zone

The material used for the reaction zone is a membrane 21 such asnitrocellulose having an average pore size of 0.45 microns (Whatman,England) and cut to 12 mm×12 mm. The membrane 21 is 0.2 mm thickpaper-backed nitrocellulose and specially treated for enhanced proteinbinding. Certified specifications given by the manufacturer (Whatman,England) include a binding capacity of 80-90 mg protein/cm², a waterflow rate of 6 mL/min/cm² and a bubble point of 3.5 bar. The reactionmembrane 21 is prepared having two immunoreactive test sites, namely atest zone and a control zone, each zone produced in the shape of adistinct vertical line. The control line and the test line arepositioned perpendicular to, but not touching, one another to provide aclear differentiation between the two. The test zone of the membrane isprepared by applying a solution of Helicobacter pylori in phosphatebuffer (pH 7 to 9.5) using a printer device (BioJet Quanti 3000dispenser). The control zone is similarly prepared by applying a mixtureof a specially calibrated antigen preparation that binds to all classesof IgG antibodies ordinarily present in a biological fluid test sampleregardless of Helicobacter pylori IgG antibody status, and thus servesas a control zone. After the membrane is dried at room temperature for10 minutes, it is treated with a solution of 1% bovine serum albumin in0.1 M sodium phosphate buffer and allowed to completely dry at ambienttemperature for approximately 24 hours.

C. Optional Spacer Layer

A spacer layer 25 supporting the reaction membrane 21 may be produced bysecuring the outer perimeter of the upper surface of the reactionmembrane 21 to the lower surface of the spacer layer 25 such that theupper surface of the reaction membrane 21 is exposed through an openingof the spacer layer 25. The upper surface of the reaction membrane 21 issealed to the lower surface of the spacer layer with a fluid-resistantadhesive so as to form an impermeable seal between the rim 27 of thespacer layer 25, defining the opening 26, and the unexposed uppersurface of the reaction membrane 21. This arrangement helps to promotethe flow of fluids in a downward, as opposed to lateral, directionthrough the reaction membrane 21 and into the absorbent pad 20 below.The spacer layer 25 may be purchased with water-soluble adhesive alreadyadhered to the lower surface, or the adhesive may be applied during themanufacturing process. The spacer layer 25 is a polystyrene materialinsert with a brown paperback double-sided tape (Halifax FoldingCompany, Nova Scotia, Canada). The reaction membrane 21 is secured tothe spacer layer 25 by the double-sided tape. The assembled spacer layer25 is approximately 29.0 mm×20.5 mm in area and 1.0 mm in thickness andis positioned on the upper surface of the absorbent pad 20 which sits inthe base of the test cartridge 14 as shown in FIGS. 4 and 5.

D. Absorbent Zone

The absorbent zone comprises a pad 20 placed directly beneath thereaction membrane 21 and securely inside the bottom member 17 of thetest cartridge 14. The pad 20 is composed of thickened compressedcellulose acetate with a porosity of 40 mL/min (Filtrona, Richmond Inc.,Richmond, Va.). It is made of synthetic fibers without the use of resinsor adhesives and provides an excellent level of aqueous fluidcompatibility. Void space is specified at 80 to 85% and absorption ofliquids at 6 times the dry unit weight and up to 90% of the total voidvolume. It is resistant to pH in the range of 2.5 to 9.5. The pad 20 isdie cut to a specification of 2.2 cm width, 3.2 cm length and 0.5 cmheight. The pad 20 fits securely into the bottom member 17 of the testcartridge 14 so as to create a compressed composite of the reactionmembrane 21 with the absorbent pad 20 to ensure a continuum of fluidcommunication between the porous materials for enhanced hydrodynamicsand complete absorption when test samples are applied to the reactionmembrane 21.

E. Dried Indicator Reagent Delivery Cap

The dried indicator reagent delivery cap 15, which houses the driedindicator reagent delivery unit 3 of the rapid test apparatus 13, iscomprised of an outer funnel sleeve 29 having an internal collar 34 atits base, an inner funnel sleeve 28 having a handle 35 extendingtherefrom, and the dried indicator reagent delivery unit 3. The funnelsleeves 28, 29 and the handle 35 are molded from a plastic material. Onepreferred plastic material is polystyrene resin (Fouzhou ChimoplusChemical Company Ltd., China). The outer 29 and inner 28 funnel sleevesare cylindrical in shape with an outside diameter of 15.0 mm and 12.5mm, respectively and the assembled cap 15 fits snugly into the reservoir22 of the test cartridge 14. The dried indicator reagent delivery cap 15is designed to be connected to the reservoir 22 of the test cartridge 14following post-application of the test sample to the reservoir 22, andremoved shortly after the multifunctional buffer has been added anddiffused through the dried indicator reagent delivery unit 3 of thefilter cap 15. In the assembled form, the volume capacity of the driedindicator reagent delivery cap 15 is about 0.5 mL.

The label zone of the dried indicator reagent delivery unit 3 iscomprised of one filter layer permeated with indicator reagent that willbe in direct fluid communication with the reaction zone of the test unit2 to improve the subsequent reactivity between the antibodies of thecolloidal gold conjugate and the antigen-coated reaction membrane 21.The filter is comprised of glass micro fiber with OVA binder (Whatman,GF/AVA) which is white and has a basis weight of 48 g/m², a thickness of0.303 mm, a flow rate of 150 s/1.5 cm, dry tensile of 640 g/1.5 cm, wettensile of 324 g/1.5 cm and a porosity of 3 sec/100 mL/in². Onceassembled, the freeze-dried colloidal gold conjugate is reconstitutedwith a solution comprising 0.1-0.15 mL of PBS buffer (0.6-0.7 mMpotassium chloride, 0.03M sodium chloride, 2-2.1 mM di-sodium hydrogenorthophosphate anhydrous, 0.3-0.4 mM potassium phosphate mono)containing 10% sugar. The colloidal gold conjugate solution is dispensed(0.1 to 0.15 mL) onto each filter and then dried at a temperature of 37to 40° C. The filter layer is die cut according to the followingspecifications: thickness, 790 to 830 microns; porosity, 1.6 to 2.0s/100mL/in²; tensile strength 14.5 N/55 mm; flow rate, 67 s/7.5 cm;absorbancy, 76.4%; pore size, 4.3 microns; wicking, 1.00 min:sec; anddiameter, 0.42 mm.

10.2 Inspection on Protein A (PA)

Formulation

Sodium Chloride

10% in DDI water

BSA

1% BSA in DDI water pH 5.00 to 9.00 (optimum 6.00).

Colloidal gold

Prepared up to the pH step.

Stock-solution of the Labeling Material

Original concentration diluted in DDI to a final concentration of 0.1-2mg/mL (optimum 0.1 mg/mL)

Procedure

-   -   Prepare a 9 serial dilution Protein A (PA).    -   To the PA, dilution add the colloidal gold already pH adjusted        in a 1:10 ratio (e.g. to 0.1 mL of PA dilution add 1 mL of        colloidal gold).    -   Incubate for 10 minutes.    -   To each dilution tubes add 8-10% of sodium chloride to a final        concentration of 1% (optimum 0.9%).    -   Incubate for 5 minutes.    -   To each tube again add 0.07-0.1% of bovine serum albumin to a        final concentration of 0.1% (optimum 0.08%).    -   Read the absorbance at 520 nm.    -   The correct concentration of protein is the minimal amount that        will inhibit flocculation.

Conc. (μg) Absorbance 1 Absorbance 2 Average 0 0.313 0.314 0.314 2 0.5240.524 0.524 3 0.533 0.532 0.533 4 0.533 0.533 0.533 5 0.540 0.540 0.5406 0.575 0.571 0.573 7 0.580 0.580 0.580 8 0.576 0.583 0.580 9 0.5760.576 576

Hughes D. A & J. E. Beesley (1998) Preparation of Colloidal Gold Probesin (ed) J. D. Pound. Methods in Molecular Biology vol 80: ImmunochemicalProtocols, 2^(nd) edition. Humana Press Inc., Totowa, N.J.

10.3 Preparation of Colloidal Gold Conjugate

Materials Sodium Citrate 0.3 mM in DDI water BSA 1% BSA in DDI water, pH5.00 to 9.00 PEG 1% polyethylene glycol (MW 15,000 to 20,000) in DDIwater, pH to 6.00 Phosphate Buffer mix 0.04M (in DI water) of NaH₂PO₄into 0.07M (in water) KH₂PO₄ in a 1:4.4 ratio; pH to 6.00 Protein A inProtein A in Hepes buffer (0.025M Hepes and Hepes Buffer 0.25 mMThimerosal pH 7.00 in ODI water) to give a final concentration on 1mg/mL. Borate buffer 0.05M of sodium borate in DDI water pH 8.50

Resuspending Buffer

8 mM di-sodium hydrogen orthophosphate anhydrous

1% bovine serum albumin

3 mM sodium azide

0.02% polyethylene glycol

0.14-0.16M sodium chloride

1.5 mM potassium dihydrogen orthophosphate

2.7 mM potassium chloride

4.3 mM tri-sodium orthophosphate

Mix the above ingredients in 1000 mL of DDI water, pH 7.30 to 7.50.

Procedure

-   -   Add 1% of tetrachloroauric acid to water for a final        concentration of 0.01%.    -   Let solution reach a hard boiling point.    -   Add 15 mL of 0.3 mM sodium citrate on a reflux for 30 minutes.    -   Remove the flask and allow the contents to cool to around 40° C.        or lower.    -   Add 60 mL of phosphate buffer (mix 0.04M (in DI water) of        NaH2PO4 into 0.07M (in water) KH2PO4 in a 1:4.4 ratio; pH to        6.00) or 50 mM of borate buffer (pH 8.50); adjust pH of the        colloidal gold to 6.00-9.00 (optimal is 6.00), if the pH is too        low, add drops of 2 mM K2CO3.    -   A portion of the solution is removed to perform an aggregation        test to know the concentration of the ligand to add to the gold        solution.    -   Add Protein A with a final concentration of 5-9 ug/mL+5%        (optimum 6+0.3=6.3 ug/mL)

$\frac{\left\lbrack {{{Final}\mspace{14mu} {PA}} + {5\%}} \right\rbrack \left( {{mg}\text{/}L} \right) \times {total}\mspace{14mu} {volume}\mspace{14mu} {in}\mspace{14mu} {flask}\mspace{14mu} (L)}{\left\lbrack {{Initial}\mspace{14mu} {PA}} \right\rbrack \left( {{mg}\text{/}{mL}} \right)}$

i.e. Total volume=500 mL CG and dH2O+15 mL sodium citrate+60 mLphosphate

-   -   buffer—3 mL test for pH=572 mL=0.572 L

$\frac{\left( {6 + 0.3} \right)\mspace{14mu} {mg}\text{/}L \times 0.572\mspace{14mu} L}{1\mspace{14mu} {mg}\text{/}{mL}} = {3.60\mspace{14mu} {mL}\mspace{14mu} {Protein}\mspace{14mu} A\mspace{14mu} {to}\mspace{14mu} {be}\mspace{14mu} {added}}$

-   -   The solution is allowed to proceed for 15-30 minutes (optimum 20        minutes).    -   The absorption of the ligand is stop by adding 10% bovine serum        albumin pH 5.00 to 9.00 final concentration of 0.1% stir for        5-15 minutes (optimum 10 minutes).    -   The labeled colloidal gold was centrifuged at a speed of 46,500        g (20,000 rpm) at a temperature of 4-5 C for 50 to 80 minutes.

Aspiration and Re-Suspension

Aspirate the supernatant with the help of a vacuum flask, taking carenot to disturb the pellet. Resuspend in re-suspending buffer (orPBS-BSA) to a final optical density of 1.180 to 4.500 (optimum 2.000) at520 nm.

Lyophilization Process

After the appropriate optical density reach, the solution was filled in0.6 mL aliquots into a 3 mL glass vials. Slotted stoppers (1-mm indiameter) were inserted halfway into the vials and transferred into thelyophilizer shelves. A temperature of −40 C was maintained for about 5hours. The primary drying was carried out at a vacuum of less than 100mTorr with a shelf temperature of −30 C for about 3 hours and acondenser temperature of less than −80 C. Followed is a shelftemperature of −10 C for about 5 hours then a shelf temperature of 0 C,vacuum of 0 mTorr for about 2 hours. A secondary drying is carry on at+20 C for about 4 hours. At the end of the process, the vials were sealunder vacuum with the slotted stoppers. The product was then removedfrom the shelves and a functional test was performed to assure thequality of the product. Samples were kept for later reference.

10.4 Stabilization of Colloidal Gold Conjugate Procedure

Prepare 1%, 2%, 5% and 10% sucrose in PBS solution, pH 7.0-7.5.

Reconstitute freeze-dried colloidal gold conjugate with a) 5 drops (150μL) b) 10 drops (350 μL) and c) 15 drops (650 μL) with each percentageof sucrose.

Apply 5 drops (150 μL) of each reconstitution to the filter medium

Result Specimen 1% 2% 5% 10% Control line 2+ 2+ 2+ 3+ Positive 1+ 1+ 1+2+/3+ control Negative neg neg neg neg control

10.5 Preparation of the Dried Indicator Reagent Delivery Unit

One of the goals in diagnostic testing is to develop a test device thatrequires few manipulative steps. Therefore, by associating the indicatorreagent with the filter medium of the dried indicator reagent deliveryunit 3, it is possible to eliminate extra steps in which the reagentsare added separately to the diagnostic device during the assay protocol.

Materials

Colloidal gold conjugate, Prepared and previously freeze-dried asdescribed above.

Sugar, e.g. trehalose, lactose, sucrose, glucose, maltose, mannose,fructose, etc.

Procedure

Reconstitute the freeze-dried colloidal gold with 0.1-0.15 mL of PBSbuffer (0.6-0.7 mM potassium chloride, 0.03M Sodium chloride, 2-2.1 mMdi-sodium hydrogen orthophosphate anhydrous, 0.3-0.4 mM potassiumphosphate mono) containing 10% sucrose.

Dispense 0.1 to 0.15 mL of colloidal gold solution onto each filter.

Let the filter dry completely at 37-40 C.

10.6 Preparation of the Multifunctional Buffer Formulation

0.01-0.1M EDTA

0.02M Sodium azide

0.05-0.1M Sodium chloride

6 mM di-sodium hydrogen orthophosphate anhydrous

0.1-0.25 mM Thimerosal

0.05-0.1% Triton® X-100

0.02-0.03M Trizma hydrochloride

0.2-0.3% Tween-20

0.5-2.5% PVP-40

Procedure

Add all ingredients together (0.01-0.1M EDTA, 0.02M sodium azide, 0.05

0.1M sodium chloride, 6 mM di-sodium hydrogen orthophosphate

anhydrous, 0.1-0.25 mM Thimerosal, 0.05-0.1% Triton® X-100, 0.02-0.03MTrizma hydrochloride, 0.2-0.3% Tween-20, 0.5-2.5% PVP-40).

Fill up with DDI water.

Adjust the pH to 7.00 to 10.00.

10.6 Assay Protocol Serum or Plasma Sample

Using a clean pipette, 1 drop of a serum or plasma sample was added tothe centre of the reaction membrane and the sample allowed to absorbcompletely through the membrane and into the absorbent material pad. Thedried indicator reagent delivery cap was connected to the reservoir ofthe test cartridge so that the dried indicator reagent delivery unit wasin fluid communication with the test unit. Ten to fifteen drops of themultifunctional buffer were subsequently added to the funnel of thedried indicator reagent delivery cap. After a brief incubation, about 1minute, during which time the resolubilized colloidal gold conjugate wasdrawn through the dried indicator reagent delivery unit, the driedindicator reagent delivery cap was removed from the test cartridge. Adistinct colored line(s), one vertical control line and one test line,developed in the centre of the reaction membrane indicating the presenceof Helicobacter pylori in the test sample. The results of the assay wererevealed in about three (3) minutes.

1. A downward or vertical flow through test device for determining thepresence or absence of a target analyte in a fluid test sample, the testdevice comprising: a test unit comprising: a reaction zone containing animmobilized capture reagent that binds a target analyte in the fluidtest sample to form a two-membered complex of a specific bindinginteraction, and an absorbent zone in vertical communication with thereaction zone, the absorbent zone comprising an absorbent materialpositioned underneath the reaction zone for facilitating the downward orvertical flow of the fluid test sample through the reaction zone andinto the absorbent zone; and a post-filter cap which is operablyaffixable to a test unit so as to be in vertical communication with areaction zone after a fluid test sample has been applied to the reactionzone, the post-filter cap comprising: an outer sleeve comprising anoutwardly extending flange, sidewalls depending from the outwardlyextending flange and terminating at an open-ended base, the base furthercomprising a collar projecting inwardly from the sidewalls; an innersleeve dimensioned to frictionally engage the inner surface of the outersleeve, the inner sleeve comprising an outwardly extending flange,sidewalls depending from the outwardly extending flange and terminatingat an open-ended base; and a removable post-filter unit supported by thecollar of the outer sleeve and held in place by the open-ended base ofthe inner sleeve, the post-filter unit comprising a porous materialhaving a direct label conjugated general marker protein complex embeddedthereon, whereby application of a buffer to the porous materialmobilizes the direct label conjugated general marker protein complex andliberates the complex from the porous material to flow-through the baseof the outer sleeve.
 2. The device according to claim 1, wherein thedirect label conjugated general marker protein is capable of binding tothe target analyte at a site which does not interfere with the specificbinding interaction between the target analyte and the capture reagent.3. The device according to claim 1, wherein the direct label conjugatedgeneral marker protein complex binds to the capture reagent at a sitewhich interferes with the specific binding interaction between thetarget analyte and the capture reagent.
 4. The device according to claim1, wherein the porous material has a pore size that allows the directlabel conjugated general marker protein complex to be effectivelyresolubilized by the buffer and transferred to the reaction zone bylaminar fluid flow.
 5. The device according to claim 4, wherein theporous material is a glass fiber material.
 6. The device according toclaim 3, wherein the direct label is colloidal gold.
 7. The deviceaccording to claim 1, wherein the general marker protein is selectedfrom the group consisting of protein A, protein G and anti-IgG.
 8. Thedevice according to claim 1, wherein the general marker protein is anantibody that binds to the target analyte in the two-membered complex.9. The device according to claim 1, wherein the general marker proteinis an antigen that binds to the target analyte in the two-memberedcomplex.
 10. The device according to claim 1, wherein the specificbinding interaction is an antibody-antigen interaction.
 11. The deviceaccording to claim 1, wherein the target analyte is an antigen and thegeneral protein marker is a monoclonal antibody or an affinity purifiedpolyclonal antibody for the antigen.
 12. The device according to claim1, wherein the reaction zone is comprised of a material which has a poresize permitting separation and filtration of unbound components from thefluid test sample and a thickness which permits an adequate amount ofcapture reagent to be immobilized thereto.
 13. The device according toclaim 1, further comprising a handle attached to the outwardly extendingflange of the outer sleeve for facilitating the attachment and removalof the post-filter cap to the test unit.
 14. The device according toclaim 13, wherein the inner space defined by the sidewalls isdimensioned to control the flow rate of the buffer through thepost-filter unit.
 15. The device according to claim 1, furthercomprising a blood separation zone in lateral communication with thereaction zone, wherein the blood separation zone has a first enddefining a region for receiving a whole blood test sample, and a secondend in communication with the reaction zone.
 16. The device according toclaim 15, wherein the blood separation zone comprises a material capableof selectively retaining cellular components of the whole blood testsample to generate a substantially cellular component-free fluid portionwhich can flow from the first end of the blood separation zone to thereaction zone.
 17. The device according to claim 16, wherein thematerial is a glass fiber matrix.
 18. The device according to claim 16,wherein the material comprises a hydrophobic carrier capable of reducingseepage of the whole blood test sample and the cellular component-freefluid portion as it migrates along the blood separation zone.
 19. Adiagnostic test kit for conducting an assay to determine the presence orabsence of a target analyte in a fluid test sample, the diagnostic testkit comprising: a test unit comprising: a reaction zone containing animmobilized capture reagent that binds a target analyte in the fluidtest sample to form a two-membered complex of a specific bindinginteraction, and an absorbent zone in vertical communication with thereaction zone, the absorbent zone comprising an absorbent materialpositioned underneath the reaction zone for facilitating the downward orvertical flow of the fluid test sample through the reaction zone andinto the absorbent zone; a post-filter cap which is operably affixableto a test unit so as to be in vertical communication with a reactionzone after a fluid test sample has been applied to the reaction zone,the post-filter cap comprising: an outer sleeve comprising an outwardlyextending flange, sidewalls depending from the outwardly extendingflange and terminating at an open-ended base, the base furthercomprising a collar projecting inwardly from the sidewalls; an innersleeve dimensioned to frictionally engage the inner surface of the outersleeve, the inner sleeve comprising an outwardly extending flange,sidewalls depending from the outwardly extending flange and terminatingat an open-ended base; and a removable post-filter unit supported by thecollar of the outer sleeve and held in place by the open-ended base ofthe inner sleeve, the post-filter unit comprising a porous materialhaving a direct label conjugated general marker protein complex embeddedthereon; and a buffer reagent for use in the assay.
 20. The diagnostictest kit according to claim 19, further comprising one or more pipettesfor use in the assay.
 21. The diagnostic test kit according to claim 19,further comprising instructions for conducting the assay.
 22. Thediagnostic test kit according to claim 19, wherein the buffer reagent isa multifunctional buffer comprising: a biological buffer to maintain thepH between 7.0 to 10.0; at least one surfactant to reduce non-specificbinding of assay reagents while simultaneously avoiding inhibition of aspecific binding interaction; a high molecular weight polymer as adispersing and suspending reagent having a molecular weight in a rangeof from about 2×10² to about 2×10⁶ D; a pH stabilizer to maintain the pHof the multifunctional buffer within a range of about pH 7.0 to 10.0; anionic salt to reduce non-specific binding of antibodies; at least onepreservative to reduce bacterial and microbial growth; and a calciumchelator to prevent a whole blood test sample from clotting; wherein thebiological buffer, the surfactant, the high molecular weight polymer,the pH stabilizer, the ionic salt, the preservative and the calciumchelator are all in effective concentrations.
 23. The kit according toclaim 19, wherein the test unit further comprises a blood separationzone in lateral communication with the reaction zone, wherein the bloodseparation zone has a first end defining a region for receiving a wholeblood test sample, and a second end in communication with the reactionzone.